Search Results (63)

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B₆-responsive seizures. To study HPP, we generated alpl^-/- zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl^-/- compared to alpl^+/+ embryos. The survival of alpl^-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl^-/- embryos. B₆ vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl^-/- embryos. Accumulation of d3-pyridoxal 5′-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl^-/- embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B₆ metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl^-/- embryos. Metabolic profiling of alpl^-/- larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B₆-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments. Full article

Background: Among abiotic stresses to agricultural crops, drought stress is the most prolific and has worldwide detrimental impacts. The soybean (Glycine max) is one of the most important sources of nutrition to both livestock and humans. Different plant introductions (PI) of soybeans have been identified to have different drought tolerance levels. Objectives: Here, two soybean lines, Pana (drought sensitive) and PI 567731 (drought tolerant) were selected to identify chemical compounds and pathways which could be targets for metabolomic analysis induced by abiotic stress. Methods: Extracts from the two lines are analyzed by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The high mass resolution and accuracy of the method allows for identification of ions from hundreds of different compounds in each cultivar. The exact m/z of these species were filtered through SoyCyc and the Human Metabolome Database to identify possible molecular formulas of the ions. Next, the exact m/z values were converted into Kendrick masses and their Kendrick mass defects (KMD) computed, which were then sorted from high to low KMD. This latter process assists in identifying many additional molecular formulas, and is noted to be particularly useful in identifying formulas whose mass difference corresponds to two hydrogen atoms. Results: In this study, more than 460 ionic formulas were identified in Pana, and more than 340 ionic formulas were identified in PI 567731, with many of these formulas reported from soybean for the first time. Conclusions: Using the SoyCyc matches, the metabolic pathways from each cultivar were compared, providing lists of molecular targets available to profile effects of abiotic stress on these soybean cultivars. Key metabolites include chlorophylls, pheophytins, mono- and diacylglycerols, cycloeucalenone, squalene, and plastoquinones and involve pathways which include the anabolism and catabolism of chlorophyll, glycolipid desaturation, and biosynthesis of phytosterols, plant sterols, and carotenoids. Full article

The growing demand for sustainable lubricant solutions is driving the exploration of bio-based materials that deliver comparable performance to conventional, primarily fossil-based lubricant chemistries. This study focuses on squalane as a sustainable base oil, which can be derived from different renewable sources. A total of two squalane products were evaluated for thermal-oxidative stability and benchmarked against a polyalphaolefin, PAO 4, of the same total carbon number. Oils artificially altered in a closed reactor were sampled and subjected to conventional lubricant analyses, including infrared spectroscopy, to determine the changes due to autoxidation over time. For in-depth information, direct-infusion high-resolution mass spectrometry and gas chromatography coupled with triple quadrupole mass spectrometry were employed to identify degradation products from thermo-oxidative stress. The results revealed substantial variability in the stability of squalane products, suggesting that differences in raw materials and production processes have a major impact on their performance, including rheological properties. The degradation products of polyalphaolefin and squalane, identified through detailed mass spectrometry, were analyzed to understand their impact on conventional physicochemical properties. While polyalphaolefin predominantly generated carboxylic acids with short to medium chain lengths as degradation products, squalane oxidation produced carboxylic acids with medium to long chain lengths as well as several alcohols and ketones. Despite these differences, squalane demonstrates its potential as a non-fossil hydrocarbon base oil, as squalane products matched and even exceeded PAO 4 stability. Full article

Background: Acute and chronic pre-traumatic cigarette smoke exposure increases morbidity and mortality after trauma and hemorrhage. In mice with a genetic deletion of the H₂S-producing enzyme cystathione-γ-lyase (CSE^−/−), providing exogenous H₂S using sodium thiosulfate (Na₂S₂O₃) improved organ function after chest trauma and hemorrhagic shock. Therefore, we evaluated the effect of Na₂S₂O₃ during resuscitation from blunt chest trauma and hemorrhagic shock on CSE^−/− mice with pre-traumatic cigarette smoke (CS) exposure. Since H₂S is well established as being able to modify energy metabolism, a specific focus was placed on whole-body metabolic pathways and mitochondrial respiratory activity. Methods: Following CS exposure, the CSE^−/− mice underwent anesthesia, surgical instrumentation, blunt chest trauma, hemorrhagic shock for over 1 h (target mean arterial pressure (MAP) ≈ 35 ± 5 mmHg), and resuscitation for up to 8 h comprising lung-protective mechanical ventilation, the re-transfusion of shed blood, fluid resuscitation, and continuous i.v. noradrenaline (NoA) to maintain an MAP ≥ 55 mmHg. At the start of the resuscitation, the mice randomly received either i.v. Na₂S₂O₃ (0.45 mg/g_bodyweight; n = 14) or the vehicle (NaCl 0.9%; n = 11). In addition to the hemodynamics, lung mechanics, gas exchange, acid–base status, and organ function, we quantified the parameters of carbohydrate, lipid, and protein metabolism using a primed continuous infusion of stable, non-radioactive, isotope-labeled substrates (gas chromatography/mass spectrometry) and the post-mortem tissue mitochondrial respiratory activity (“high-resolution respirometry”). Results: While the hemodynamics and NoA infusion rates did not differ, Na₂S₂O₃ was associated with a trend towards lower static lung compliance (p = 0.071) and arterial PO₂ (p = 0.089) at the end of the experiment. The direct, aerobic glucose oxidation rate was higher (p = 0.041) in the Na₂S₂O₃-treated mice, which resulted in lower glycemia levels (p = 0.050) and a higher whole-body CO₂ production rate (p = 0.065). The mitochondrial respiration in the heart, kidney, and liver tissue did not differ. While the kidney function was comparable, the Na₂S₂O₃-treated mice showed a trend towards a shorter survival time (p = 0.068). Conclusions: During resuscitation from blunt chest trauma and hemorrhagic shock in CSE^−/− mice with pre-traumatic CS exposure, Na₂S₂O₃ was associated with increased direct, aerobic glucose oxidation, suggesting a switch in energy metabolism towards preferential carbohydrate utilization. Nevertheless, treatment with Na₂S₂O₃ coincided with a trend towards worsened lung mechanics and gas exchange, and, ultimately, shorter survival. Full article

Background/Objectives: Profiling of metabolites and lipids in biological samples can provide invaluable insights into life-sustaining chemical processes. The ability to detect both metabolites and lipids in the same sample can enhance these understandings and connect cellular dynamics. However, simultaneous detection of metabolites and lipids is generally hampered by chromatographic systems tailored to one molecular type. This void can be filled by direct infusion mass spectrometry (MS), where all ionizable molecules can be detected simultaneously. However, in direct infusion MS, the high chemical complexity of biological samples can introduce limitations in detectability due to matrix effects causing ionization suppression. Methods: Decreased sample complexity and increased detectability and molecular coverage was provided by combining our direct infusion probe (DIP) with liquid–liquid extraction (LLE) and directly sampling the different phases for direct infusion. Three commonly used LLE methods for separating lipids and metabolites were evaluated. Results: The butanol–methanol (BUME) method was found to be preferred since it provides high molecular coverage and have low solvent toxicity. The established BUME DIP-MS method was used as a fast and sensitive analysis tool to study chemical changes in insulin-secreting cells upon glucose stimulation. By analyzing the metabolome at distinct time points, down to 1-min apart, we found high dynamics of the intracellular metabolome. Conclusions: The rapid workflow with LLE DIP-MS enables higher sensitivity of phase separated metabolites and lipids. The application of BUME DIP-MS provides novel information on the dynamics of the intracellular metabolome of INS-1 during the two phases of insulin release for both metabolite and lipid classes. Full article

This study presents a detailed analysis of the thermal degradation and kinetic behavior of two Amazonian wood species, Goupia glabra (cupiúba) and Manilkara huberi (maçaranduba), using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR-ATR), and direct infusion mass spectrometry (DIMS). Wood samples were subjected to controlled heating rates of 20, 40, and 60 °C/min from 25 to 800 °C under an argon atmosphere. TGA revealed moisture evaporation below 120 °C, with hemicellulose degradation occurring between 220 and 315 °C, cellulose decomposition between 315 and 400 °C, and lignin breakdown over a broader range from 180 to 900 °C. The highest rate of mass loss occurred at 363.99 °C for G. glabra and 360.27 °C for M. huberi at a heating rate of 20 °C/min, with shifts to higher temperatures at faster heating rates. Activation energies were calculated using Arrhenius and Kissinger models, yielding values between 53.46–61.45 kJ/mol for G. glabra and 58.18–62.77 kJ/mol for M. huberi, confirming their stable thermal profiles. DSC analysis identified a significant endothermic peak related to moisture evaporation below 100 °C, followed by two exothermic peaks. For G. glabra, the first exothermic peak appeared at 331.45 °C and the second at 466.08 °C, while for M. huberi, these occurred at 366.41 °C and 466.08 °C, indicating the decomposition of hemicellulose, cellulose, and lignin. Enthalpy values for G. glabra were 12,633.37 mJ and 18,652.66 mJ for the first and second peaks, respectively, while M. huberi showed lower enthalpies of 9648.04 mJ and 14,417.68 mJ, suggesting a higher energy release in G. glabra. FTIR-ATR analysis highlighted the presence of key functional groups in both species, with strong absorption bands in the 3330–3500 cm⁻¹ region corresponding to O-H stretching vibrations, indicative of hydroxyl groups in cellulose and hemicellulose. The 1500–1600 cm⁻¹ region, representing aromatic C=C vibrations, confirmed the presence of lignin. Quantitatively, these results suggest a high content of cellulose and lignin in both species. DIMS analysis further identified polyphenolic compounds and triterpenoids in M. huberi, with major ions at m/z 289 and 409, while G. glabra showed steroidal and polyphenolic compounds with a base peak at m/z 395. These findings indicate the significant presence of bioactive compounds, contributing to the wood’s resistance to microbial degradation. This comprehensive thermal and chemical characterization suggests that both species have potential industrial applications in environments requiring high thermal stability. Full article

The acrylation degree of vegetable oils plays a relevant role in determining the mechanical properties of the resulting polymers. Both epoxide and acrylate functionalities participate in polymerization reactions, producing various types of chemical bonds in the polymer network, which contribute to specific properties such as molecular size distribution, crosslinking degree, and glass transition temperature (Tg). The accurate identification of epoxide and acrylated groups in triglyceride molecules helps to predict their behavior during the polymerization process. A methodology based on analytical spectrometric techniques, such as direct infusion, mass spectrometry with electrospray ionization, and ultra-high-performance liquid chromatography, is used in combination with FTIR and ¹H NMR to characterize the epoxy and acrylic functionalities in the fatty chains with different numbers of carbon atoms of partially acrylated triglycerides obtained by a non-catalytic reaction. Full article

Most clinical isolates of both Staphylococcus aureus and Staphylococcus epidermidis show the capacity to adhere to abiotic surfaces and to develop biofilms resulting in a contribution to chronic human skin infections. Antibiotic resistance and poor biofilm penetration are the main causes of ineffective therapeutic treatment in killing bacteria within biofilms. A possible strategy could be represented by drug delivery systems, such as nanoemulsions (composed of bioactive oil, surfactant and water phase), which are useful for enhancing the drug permeation of a loaded drug inside the biofilm and its activity. Phytochemical characterization of Pistacia lentiscus oil (LO) by direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) allowed the identification of bioactive compounds with antimicrobial properties, including fatty acids and phenolic compounds. Several monoterpenes and sesquiterpenes have been also detected and confirmed by gas chromatography–mass spectrometric (GC-MS) analysis, together providing a complete metabolomic profiling of LO. In the present study, a nanoemulsion composed of LO has been employed for improving Levofloxacin water solubility. A deep physical–chemical characterization of the nanoemulsion including hydrodynamic diameter, ζ-potential, morphology, entrapment efficiency, stability release and permeation studies was performed. Additionally, the antimicrobial/antibiofilm activity of these preparations was evaluated against reference and clinical Staphylococcus spp. strains. In comparison to the free-form antibiotic, the loaded NE nanocarriers exhibited enhanced antimicrobial activity against the sessile forms of Staphylococcus spp. strains. Full article

Direct infusion–high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of ¹³C and/or ¹⁵N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics. Full article

Ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to analyze a colorant and silk, which were prepared and dyed using shellfish (Hexaplex trunculus L.) purple. Solutions of colorant and silk extracts were analyzed immediately after preparation (fresh samples) and after storing them in the dark for thirty days (aged sample I). Moreover, a silk sample was subjected to artificially accelerated ageing under UV radiation (aged sample II). The application of the UHPLC-MS/MS method leads to the detection of (i) the major coloring components of shellfish purple, which are indigotin, indirubin, 6-bromoindigotin, 6′-bromoindirubin, 6-bromoindirubin, 6,6′-dibromoindigotin, 6,6′-dibromoindirubin; (ii) four minor indigoid components in shellfish purple (compounds A, B, C and D), which belong to the same structural class as indirubin, and whose identification has been reported only once in the past; and (iii) eight degradation products (isatin, degradation products DP3, DP4, DP5, DP6, DP7, DP9 and DP10). The latter were also detected in stored indigotin solution, except for DP 6, which was used as reference sample. The method development was assisted by a new solution preparation approach for investigating compound fragmentation, using a solvent system compatible with direct infusion ESI. This system replaceddimethyl sulfoxide, which inhibits detection through electrospray ionization. Full article

Lipids represent a large group of biomolecules that are responsible for various functions in organisms. Diseases such as diabetes, chronic inflammation, neurological disorders, or neurodegenerative and cardiovascular diseases can be caused by lipid imbalance. Due to the different stereochemical properties and composition of fatty acyl groups of molecules in most lipid classes, quantification of lipids and development of lipidomic analytical techniques are problematic. Identification of different lipid species from complex matrices is difficult, and therefore individual analytical steps, which include extraction, separation, and detection of lipids, must be chosen properly. This review critically documents recent strategies for lipid analysis from sample pretreatment to instrumental analysis and data interpretation published in the last five years (2019 to 2023). The advantages and disadvantages of various extraction methods are covered. The instrumental analysis step comprises methods for lipid identification and quantification. Mass spectrometry (MS) is the most used technique in lipid analysis, which can be performed by direct infusion MS approach or in combination with suitable separation techniques such as liquid chromatography or gas chromatography. Special attention is also given to the correct evaluation and interpretation of the data obtained from the lipid analyses. Only accurate, precise, robust and reliable analytical strategies are able to bring complex and useful lipidomic information, which may contribute to clarification of some diseases at the molecular level, and may be used as putative biomarkers and/or therapeutic targets. Full article

Background: An accurate measurement of the glomerular filtration rate (GFR) is essential for detecting renal insufficiency in living kidney donors. Iohexol is a “near-ideal” exogenous filtration marker for GFR measurements that has attracted increasing interest in clinical practice because it is non-toxic, non-radioactive, readily available, and easy to measure. In this study, we aimed to set up a laboratory test to conveniently assess the plasma clearance of iohexol in living kidney donors. Methods: A workflow was established in the institution’s infusion clinic to administer iohexol and to collect three timed blood samples from renal transplant donors. Iohexol was thereafter measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The serum proteins were precipitated and the supernatant containing iohexol was diluted prior to the LC-MS/MS analysis. The LC-MS/MS method was developed on a Thermo Vanquish UHPLC coupled with a TSQ Endura triple quadruple mass spectrometer with a total run time of 2.5 min. The analytical performance of the method was assessed. Results: The LC-MS/MS method demonstrated a good analytical performance. To calculate the iohexol clearance rate and the GFR, automated data integration and a result calculation were accomplished by using a custom Python script. Automated result reporting was achieved using a laboratory informatics system (LIS) vendor’s direct media interface. Conclusions: We developed and implemented a laboratory test to assess the plasma clearance of iohexol. A workflow was established in the hospital to reliably measure the GFR in living kidney donors, with a potential to be further expanded into other areas where an accurate GFR measurement is needed. Full article

Direct infusion nanoelectrospray high-resolution mass spectrometry (DI-nESI-HRMS) is a promising tool for high-throughput metabolomics analysis. However, metabolite assignment is limited by the inadequate mass accuracy and chemical space of the metabolome database. Here, a serum metabolome characterization method was proposed to make full use of the potential of DI-nESI-HRMS. Different from the widely used database search approach, unambiguous formula assignments were achieved by a reaction network combined with mass accuracy and isotopic patterns filter. To provide enough initial known nodes, an initial network was directly constructed by known metabolite formulas. Then experimental formula candidates were screened by the predefined reaction with the network. The effects of sources and scales of networks on assignment performance were investigated. Further, a scoring rule for filtering unambiguous formula candidates was proposed. The developed approach was validated by a pooled serum sample spiked with reference standards. The coverage and accuracy rates for the spiked standards were 98.9% and 93.6%, respectively. A total of 1958 monoisotopic features were assigned with unique formula candidates for the pooled serum, which is twice more than the database search. Finally, a case study of serum metabolomics in diabetes was carried out using the developed method. Full article

Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disease, commonly caused by pathogenic MYBPC3 variants, and a significant cause of sudden cardiac death. Severity is highly variable, with incomplete penetrance among genotype-positive family members. Previous studies demonstrated metabolic changes in HCM. We aimed to identify metabolite profiles associated with disease severity in carriers of MYBPC3 founder variants using direct-infusion high-resolution mass spectrometry in plasma of 30 carriers with a severe phenotype (maximum wall thickness ≥20 mm, septal reduction therapy, congestive heart failure, left ventricular ejection fraction <50%, or malignant ventricular arrhythmia) and 30 age- and sex-matched carriers with no or a mild phenotype. Of the top 25 mass spectrometry peaks selected by sparse partial least squares discriminant analysis, XGBoost gradient boosted trees, and Lasso logistic regression (42 total), 36 associated with severe HCM at a p < 0.05, 20 at p < 0.01, and 3 at p < 0.001. These peaks could be clustered to several metabolic pathways, including acylcarnitine, histidine, lysine, purine and steroid hormone metabolism, and proteolysis. In conclusion, this exploratory case-control study identified metabolites associated with severe phenotypes in MYBPC3 founder variant carriers. Future studies should assess whether these biomarkers contribute to HCM pathogenesis and evaluate their contribution to risk stratification. Full article

In metabolomics, many metabolites are measured simultaneously in a single run. Such analytical performance opens up prospects for clinical laboratory diagnostics. In this work, a mass spectrometric metabogram was developed as a simplified and clinically applicable way of measuring the blood plasma metabolome. To develop the metabogram, blood plasma samples from healthy male volunteers (n = 48) of approximately the same age, direct infusion mass spectrometry (DIMS) of the low molecular fraction of samples, and principal component analysis (PCA) of the mass spectra were used. The seven components of the metabogram defined by PCA, which cover ~70% of blood plasma metabolome variability, were characterized using a metabolite set enrichment analysis (MSEA) and clinical test results of participating volunteers. It has been established that the components of the metabogram are functionally related groups of the blood metabolome associated with regulation, lipid–carbohydrate, and lipid–amine blood components, eicosanoids, lipid intake into the organism, and liver function thereby providing a lot of clinically relevant information. Therefore, metabogram provides the possibility to apply the metabolomics performance in the clinic. The features of the metabogram are also discussed in comparison with the thin-layer chromatography and with the analysis of blood metabolome by liquid chromatography combined with mass spectrometry. Full article