Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of TLC, chemical staining, carbohydrate-recognized ligand-binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4) and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches.

Robust and high-throughput lipidomic quantitation of human blood samples using flow injection analysis with tandem mass spectrometry for clinical use.

Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7 min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles.

Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2-µm particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full-scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full-scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS-based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

Simple and Reproducible Derivatization with Benzoyl Chloride: Improvement of Sensitivity for Multiple Lipid Classes in RP-UHPLC/MS.

The chemical derivatization of multiple lipid classes was developed using benzoyl chloride as a nonhazardous derivatization agent at ambient conditions. The derivatization procedure was optimized with standards for 4 nonpolar and 8 polar lipid classes and measured by reversed-phase ultrahigh-performance liquid chromatography-tandem mass spectrometry. The derivatization and nonderivatization approaches were compared on the basis of the calibration curves of 22 internal standards from 12 lipid classes. The new method decreased the limit of detection 9-fold for monoacylglycerols (0.9-1.0 nmol/mL), 6.5-fold for sphingoid base (0.2 nmol/mL), and 3-fold for diacylglycerols (0.9 nmol/mL). The sensitivity expressed by the ratio of calibration slopes was increased 2- to 10-fold for almost all investigated lipid classes and even more than 100-fold for monoacylglycerols. Moreover, the benzoylation reaction produces a more stable derivative of cholesterol in comparison to the easily in-source fragmented nonderivatized form and enabled the detection of fatty acids in a positive ion mode, which does not require polarity switching as for the nonderivatized form. The intralaboratory comparison with an additional operator without previous derivatization experiences shows the simplicity, robustness, and reproducibility. The stability of the derivatives was determined by periodical measurements during a one month period and five freeze/thaw cycles. The fully optimized derivatization method was applied to human plasma, which allows the detection of 169 lipid species from 11 lipid classes using the high confidence level of identification in reversed-phase (RP)-ultra high performance liquid chromatography (UHPLC)/mass spectrometry (MS). Generally, we detected more lipid species for monoacylglycerols, diacylglycerols, and sphingoid bases in comparison with previously reported papers without the derivatization.

Cadmium Isotope Fractionation during Complexation with Humic Acid.

Cadmium (Cd) isotopes are known to fractionate during complexation with various environmentally relevant surfaces and ligands. Our results, which were obtained using (i) batch experiments at different Cd concentrations, ionic strengths, and pH values, (ii) modeling, and (iii) infrared and X-ray absorption spectroscopies, highlight the preferential enrichment of light Cd isotopes bound to humic acid (HA), leaving the heavier Cd pool preferentially in solution (Δ114/110CdHA-Cd(aq) of -0.15 ± 0.01‰). At high ionic strengths, Cd isotope fractionation mainly depends on its complexation with carboxylic sites. Outer-sphere complexation occurs at equilibrium together with inner-sphere complexation as well as with the change of the first Cd coordination and its hydration complexes in solution. At low ionic strengths, nonspecific Cd binding induced by electrostatic attractions plays a dominant role and promotes Cd isotope fractionation during complexation. This significant outcome elucidates the mechanisms involved in HA-Cd interactions. The results can be used during (i) fingerprinting the available Cd in soil solution after its complexation with solid or soluble natural organic matter and (ii) evaluating the contribution of Cd complexation with organic ligands and phytoplankton-derived debris versus Cd assimilation by phytoplankton in seawater.

Evaluating the effectiveness of sulfidated nano zerovalent iron and sludge co-application for reducing metal mobility in contaminated soil.

Sewage sludge has long been applied to soils as a fertilizer yet may be enriched with leachable metal(loid)s and other pollutants. Sulfidated nanoscale zerovalent iron (S-nZVI) has proven effective at metal sorption; however, risks associated with the use of engineered nanoparticles cannot be neglected. This study investigated the effects of the co-application of composted sewage sludge with S-nZVI for the stabilization of Cd, Pb, Fe, Zn. Five treatments (control, Fe grit, composted sludge, S-nZVI, composted sludge and S-nZVI), two leaching fluids; synthetic precipitation leaching procedure (SPLP) and toxicity characteristic leaching procedure (TCLP) fluid were used, samples were incubated at different time intervals of 1 week, 1, 3, and 6 months. Fe grit proved most efficient in reducing the concentration of extractable metals in the batch experiment; the mixture of composted sludge and S-nZVI was the most effective in reducing the leachability of metals in the column systems, while S-nZVI was the most efficient for reducing about 80% of Zn concentration in soil solution. Thus, the combination of two amendments, S-nZVI incorporated with composted sewage sludge and Fe grit proved most effective at reducing metal leaching and possibly lowering the associated risks. Future work should investigate the longer-term efficiency of this combination.

HILIC/MS quantitation of low-abundant phospholipids and sphingolipids in human plasma and serum: Dysregulation in pancreatic cancer.

A new hydrophilic interaction liquid chromatography - mass spectrometry method is developed for low-abundant phospholipids and sphingolipids in human plasma and serum. The optimized method involves the Cogent Silica type C hydride column, the simple sample preparation by protein precipitation, and the removal of highly abundant lipid classes using the postcolumn valve directed to waste during two elution windows. The method allows a highly confident and sensitive identification of low-abundant lipid classes in human plasma (246 lipid species from 24 lipid subclasses) based on mass accuracy and retention dependencies in both polarity modes. The method is validated for quantitation using two internal standards (if available) for each lipid class and applied to human plasma and serum samples obtained from patients with pancreatic ductal adenocarcinoma (PDAC), healthy controls, and NIST SRM 1950. Multivariate data analysis followed by various statistical projection methods is used to determine the most dysregulated lipids. Significant downregulation is observed for lysophospholipids with fatty acyl composition 16:0, 18:0, 18:1, and 18:2. Distinct trends are observed for phosphatidylethanolamines (PE) in relation to the bonding type of fatty acyls, where most PE with acyl bonds are upregulated, while ether/plasmenyl PE are downregulated. For the sphingolipid category, sphingolipids with very long N-acyl chains are downregulated, while sphingolipids with shorter N-acyl chains were upregulated in PDAC. These changes are consistently observed for various classes of sphingolipids, ranging from ceramides to glycosphingolipids, indicating a possible metabolic disorder in ceramide biosynthesis caused by PDAC.

Nano zerovalent Fe did not reduce metal(loid) leaching and ecotoxicity further than conventional Fe grit in contrasting smelter impacted soils: A 1-year field study.

The majority of the studies on nanoscale zero-valent iron (nZVI) are conducted at a laboratory-scale, while field-scale evidence is scarce. The objective of this study was to compare the metal(loid) immobilization efficiency of selected Fe-based materials under field conditions for a period of one year. Two contrasting metal(loid) (As, Cd, Pb, Zn) enriched soils from a smelter-contaminated area were amended with sulfidized nZVI (S-nZVI) solely or combined with thermally stabilized sewage sludge and compared to amendment with microscale iron grit. In the soil with higher pH (7.5) and organic matter content (TOC = 12.7 %), the application of amendments resulted in a moderate increase in pH and reduced As, Cd, Pb, and Zn leaching after 1-year, with S-nZVI and sludge combined being the most efficient, followed by iron grit and S-nZVI alone. However, the amendments had adverse impacts on microbial biomass quantity, S-nZVI being the least damaging. In the soil with a lower pH (6.0) and organic matter content (TOC = 2.3 %), the results were mixed; 0.01 M CaCl2 extraction data showed only S-nZVI with sludge as remaining effective in reducing extractable concentrations of metals; on the other hand, Cd and Zn concentrations were increased in the extracted soil pore water solutions, in contrast to the two conventional amendments. Despite that, S-nZVI with sludge enhanced the quantity of microbial biomass in this soil. Additional earthworm avoidance data indicated that they generally avoided soil treated with all Fe-based materials, but the presence of sludge impacted their preferences somewhat. In summary, no significant differences between S-nZVI and iron grit were observed for metal(loid) immobilization, though sludge significantly improved the performance of S-nZVI in terms of soil health indicators. Therefore, this study indicates that S-nZVI amendment of soils alone should be avoided, though further field evidence from a broader range of soils is now required.

Lipidomic and metabolomic analysis reveals changes in biochemical pathways for non-small cell lung cancer tissues.

Lung cancer represents one of the leading worldwide causes of cancer death, but the pathobiochemistry of this disease is still not fully understood. Here we characterize the lipidomic and metabolomic profiles of the tumor and surrounding normal tissues for 23 patients with non-small cell lung cancer. In total, 500 molecular species were identified and quantified by a combination of the lipidomic shotgun tandem mass spectrometry (MS/MS) analysis and the targeted metabolomic approach using liquid chromatography (LC) - MS/MS. The statistical evaluation includes multivariate and univariate methods with the emphasis on paired statistical approaches. Our research revealed significant changes in several biochemical pathways related to the central carbon metabolism, acylcarnitines, dipeptides as well as the disruption in the lipid metabolism observed mainly for glycerophospholipids, sphingolipids, and cholesteryl esters.

Removal and transformation of sulfamethoxazole in acclimated biofilters with various operation modes - Implications for full-scale application.

The knowledge gaps regarding the degradation of sulfamethoxazole (SMX) in biofilters include the effect of aeration, constant feeding with readily biodegradable organic carbon and the presence of reactive media such as manganese oxides (MnOx). Thus, the goal of this study was to assess the removal of SMX in lab-scale biofilters with various operation variables: aeration, presence of MnOx as an amendment of filtering medium and the presence of readily biodegradable organic carbon (acetate). The sand used in the experiment as a filtering medium was previously exposed to the presence of SMX and acetate, which provided acclimation of the biomass. The removal of SMX was complete (>99%) with the exception of the unaerated columns fed with the influent containing acetate, due to apparent slower rate of SMX degradation. The obtained results suggest that bacteria were able to degrade SMX as a primary substrate and the degradation of this compound was subsequent to the depletion of acetate. The LC-MS/MS analysis of the effluents indicated several biotransformation reactions for SMX: (di)hydroxylation, acetylation, nitrosation, deamonification, S-N bond cleavage and isoxazole-ring cleavage. The relative abundance of transformation products was decreased in the presence of MnOx or acetate. Based on the Microtox assay, only the effluents from the unaerated columns filled with MnOx were classified as non-toxic. The results offer important implications for the design of biofilters for the elimination of SMX, namely that biofilters offer the greatest performance when fed with secondary wastewater and operated as non-aerated systems with a filtering medium containing MnOx.

Plasma lipidomic profiles of kidney, breast and prostate cancer patients differ from healthy controls.

Early detection of cancer is one of the unmet needs in clinical medicine. Peripheral blood analysis is a preferred method for efficient population screening, because blood collection is well embedded in clinical practice and minimally invasive for patients. Lipids are important biomolecules, and variations in lipid concentrations can reflect pathological disorders. Lipidomic profiling of human plasma by the coupling of ultrahigh-performance supercritical fluid chromatography and mass spectrometry is investigated with the aim to distinguish patients with breast, kidney, and prostate cancers from healthy controls. The mean sensitivity, specificity, and accuracy of the lipid profiling approach were 85%, 95%, and 92% for kidney cancer; 91%, 97%, and 94% for breast cancer; and 87%, 95%, and 92% for prostate cancer. No association of statistical models with tumor stage is observed. The statistically most significant lipid species for the differentiation of cancer types studied are CE 16:0, Cer 42:1, LPC 18:2, PC 36:2, PC 36:3, SM 32:1, and SM 41:1 These seven lipids represent a potential biomarker panel for kidney, breast, and prostate cancer screening, but a further verification step in a prospective study has to be performed to verify clinical utility.