Ultra-high Pressure Liquid Chromatography-mass Spectrometry Research Articles

An integrated metabo-lipidomics profile of induced sputum for the identification of novel biomarkers in the differential diagnosis of asthma and COPD

BackgroundDue to their complexity and to the presence of common clinical features, differentiation between asthma and chronic obstructive pulmonary disease (COPD) can be a challenging task, complicated in such cases also by asthma–COPD overlap syndrome. The distinct immune/inflammatory and structural substrates of COPD and asthma are responsible for significant differences in the responses to standard pharmacologic treatments. Therefore, an accurate diagnosis is of central relevance to assure the appropriate therapeutic intervention in order to achieve safe and effective patient care. Induced sputum (IS) accurately mirrors inflammation in the airways, providing a more direct picture of lung cell metabolism in comparison to those specimen that reflect analytes in the systemic circulation.MethodsAn integrated untargeted metabolomics and lipidomics analysis was performed in IS of asthmatic (n = 15) and COPD (n = 22) patients based on Ultra-High-Pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and UHPLC–tandem MS (UHPLC-MS/MS). Partial Least Squares-Discriminant Analysis (PLS-DA) was applied to resulting dataset. The analysis of main enriched metabolic pathways and the association of the preliminary metabolites/lipids pattern identified to clinical parameters of asthma/COPD differentiation were explored. Multivariate ROC analysis was performed in order to determine the discriminatory power and the reliability of the putative biomarkers for diagnosis between COPD and asthma.ResultsPLS-DA indicated a clear separation between COPD and asthmatic patients. Among the 15 selected candidate biomarkers based on Variable Importance in Projection scores, putrescine showed the highest score. A differential IS bio-signature of 22 metabolites and lipids was found, which showed statistically significant variations between asthma and COPD. Of these 22 compounds, 18 were decreased and 4 increased in COPD compared to asthmatic patients. The IS levels of Phosphatidylethanolamine (PE) (34:1), Phosphatidylglycerol (PG) (18:1;18:2) and spermine were significantly higher in asthmatic subjects compared to COPD.ConclusionsThis is the first pilot study to analyse the IS metabolomics/lipidomics signatures relevant in discriminating asthma vs COPD. The role of polyamines, of 6-Hydroxykynurenic acid and of d-rhamnose as well as of other important players related to the alteration of glycerophospholipid, aminoacid/biotin and energy metabolism provided the construction of a diagnostic model that, if validated on a larger prospective cohort, might be used to rapidly and accurately discriminate asthma from COPD.Graphical

Evaluation of Carbonized Corncobs for Removal of Microcystins and Nodularin-R from Water

Microcystins (MCs) and nodularins (NODs) are cyanotoxins that can be found in water bodies during cyanobacterial harmful algal blooms (cyanoHABs). Consumption of water contaminated with cyanotoxins leads to health risks for humans and animals. Herein, corncob-based biochar and activated carbon (AC) were initially investigated for the sorption of six common MC congeners (MC-RR, MC-YR, MC-LR, MC-LA, MC-LW, and MC-LF) and nodularin-R (NOD-R) from spiked water. Biochar was prepared by refluxing commercial corncob with HCl and heating it to 250, 300, or 350 °C. AC was prepared by chemical activation of corncob with H3PO4 at 500 °C under a nitrogen atmosphere. Low-temperature nitrogen adsorption measurements confirmed that H3PO4-AC has a higher specific surface area (≈1100 m2/g) and total pore volume (≈0.75 cm3/g) than biochar and commercial AC. H3PO4-AC showed the maximum efficacy, among all corncob-based sorbents, to remove MCs and NOD-R from water as confirmed by experiments that involved sample analyses by ultrahigh-pressure liquid chromatography-mass spectrometry (UHPLC-MS). The effect of natural organic matter (NOM) on the adsorption of MCs was checked by incubating sorbents with Lake Erie water collected during cyanoHABs from 2020 to 2022. The total concentration (extracellular and intracellular) of studied MC congeners ranged from 1.37 µg/L to 438.51 µg/L and 50 mg of H3PO4-AC completely removed them from 3 mL of lake water. The effect of water pH on cyanotoxin adsorption was studied at pH values of 5.5, 7.0, and 8.5 at both a lower (10 μg/L each) and a higher (50 μg/L each) toxin concentration. Removal was influenced by solution pH at both concentrations when using biochar, while only at higher toxin concentration when using H3PO4-AC. At higher MC and NOD-R concentrations, competitive adsorption was prominent, and overall, the adsorption increased at acidic pH (5.5). The study results suggest that processed corncobs can remove a significant amount of MCs and NOD-R from water, and the measured sorption capacity of H3PO4-AC was ~20 mg of MC-LR and NOD-R per g of this sorbent.

Predicting effect of anti-PD-1/PD-L1 inhibitors therapy for hepatocellular carcinoma by detecting plasma metabolite based on UHPLC-MS.

Anti-PD-1/PD-L1 inhibitors therapy has become a promising treatment for hepatocellular carcinoma (HCC), while the therapeutic efficacy varies significantly among effects for individual patients are significant difference. Unfortunately, specific predictive biomarkers indicating the degree of benefit for patients and thus guiding the selection of suitable candidates for immune therapy remain elusive.no specific predictive biomarkers are available indicating the degree of benefit for patients and thus screening the preferred population suitable for the immune therapy. Ultra-high-pressure liquid chromatography-mass spectrometry (UHPLC-MS) considered is an important method for analyzing biological samples, since it has the advantages of high rapid, high sensitivity, and high specificity. Ultra-high-pressure liquid chromatography-mass spectrometry (UHPLC-MS) has emerged as a pivotal method for analyzing biological samples due to its inherent advantages of rapidity, sensitivity, and specificity. In this study, potential metabolite biomarkers that can predict the therapeutic effect of HCC patients receiving immune therapy were identified by UHPLC-MS. A partial least-squares discriminant analysis (PLS-DA) model was established using 14 glycerophospholipid metabolites mentioned above, and good prediction parameters (R2 = 0.823, Q2 = 0.615, prediction accuracy = 0.880 and p < 0.001) were obtained. The relative abundance of glycerophospholipid metabolite ions is closely related to the survival benefit of HCC patients who received immune therapy. This study reveals that glycerophospholipid metabolites play a crucial role in predicting the efficacy of immune therapy for HCC.

Clinical pharmacokinetic evaluation of Withania somnifera (L.) Dunal root extract in healthy human volunteers: A non-randomized, single dose study utilizing UHPLC-MS/MS analysis

Ethnopharmacological relevanceWithania somnifera (L.) Dunal; (Solanaceae), commonly known as Ashwagandha, is one of the most significant medicinal herbs in ‘Ayurveda’, a traditional Indian medicine used for centuries with evidence in scriptures. Ashwagandha was mentioned in old Ayurvedic medical literature such as Charaka Samhita and Sushruta Samhita for improving weight and strength, with multiple citations for internal and exterior usage in emaciation and nourishing the body. Ethnopharmacological evidence revealed that it was used to relieve inflammation, reduce abdominal swelling, as a mild purgative, and treat swollen glands. The root was regarded as a tonic, aphrodisiac, and emmenagogue in the Unani tradition of the Indian medicinal system. Further, Ashwagandha has been also described as an Ayurvedic medicinal plant in the Ayurvedic Pharmacopoeia of India extending informed therapeutic usage and formulations. Despite the widespread ethnopharmacological usage of Ashwagandha, clinical pharmacokinetic parameters are lacking in the literature; hence, the findings of this study will be relevant for calculating doses for future clinical evaluations of Ashwagandha root extract. AimThis study aimed to develop a validated and highly sensitive bioanalytical method for quantifying withanosides and withanolides of the Ashwagandha root extract in human plasma to explore its bioaccessibility. Further to apply a developed method to perform pharmacokinetics of standardized Withania somnifera (L.) Dunal root extract (WSE; AgeVel®/Witholytin®) capsules in healthy human volunteers. MethodsA sensitive, reliable, and specific ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous quantification of five major withanosides and withanolides (withanoside IV, withanoside V, withanolide A, withaferin A, and 12-deoxy-withastramonolide) in human plasma. Further for the study, eighteen healthy male volunteers (18–45 years) were enrolled in a non-randomized, open-label, single period, single treatment, clinical pharmacokinetic study and given a single dose (500 mg) of WSE (AgeVel®/Witholytin®) capsules containing not less than 7.5 mg of total withanolides under fasting condition. Later, pharmacokinetic profiles were assessed using the plasma concentration of each bioactive constituent Vs. time data. ResultsFor all five constituents, the bioanalytical method demonstrated high selectivity, specificity, and linearity. There was no carryover, and no matrix effect was observed. Furthermore, the inter-day and intra-day precision and accuracy results fulfilled the acceptance criteria. Upon oral administration of WSE capsules, Cmax was found to be 0.639 ± 0.211, 2.926 ± 1.317, 2.833 ± 0.981, and 5.498 ± 1.986 ng/mL for withanoside IV, withanolide A, withaferin A, and 12-deoxy-withastramonolide with Tmax of 1.639 ± 0.993, 1.361 ± 0.850, 0.903 ± 0.273, and 1.375 ± 0.510 h respectively. Further, withanoside V was also detected in plasma; but its concentration was found below LLOQ. ConclusionThe novel and first-time developed bioanalytical method was successfully applied for the quantification of five bio-active constituents in human volunteers following administration of WSE capsules, indicating that withanosides and withanolides were rapidly absorbed from the stomach, have high oral bioavailability, and an optimum half-life to produce significant pharmacological activity. Further, AgeVel®/Witholytin® was found safe and well tolerated after oral administration, with no adverse reaction observed at a 500 mg dose.

DDDR-14. DEVELOPMENT OF A NOVEL CYP17A1-TARGETED INHIBITOR FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a highly aggressive and invasive brain tumor, resistant to current standard treatments, including temozolomide (TMZ) and radiotherapy. Despite these interventions, GBM's intrinsic drug resistance often renders it incurable, thus highlighting the dire need for novel therapeutic strategies. In this study, we used ultra-high pressure liquid chromatography-mass spectrometry to examine the levels of neurosteroids in tumor specimens from primary and recurrent GBM patients. This led to the discovery of differential expression patterns, most notably in androgen, including dehydroepiandrosterone and testosterone. Our results indicated androgen over-production weakens the GBM's sensitivity to TMZ by inducing post-translational modification of Sp1 protein, which aids in DNA repair. Interestingly, Sp1 activity is further enhanced by histone deacetylase (HDAC) 6, which in turn diminishes the DNA damage caused by TMZ. In addition, proteomic analysis revealed an overexpression of cytochrome (CYP) 17A1, particularly in recurrent GBM, which led us to hypothesize that a drug inhibiting both CYP17A1 and HDAC6 could potentially serve as a viable treatment for TMZ-resistant GBM. To validate this, the drug was tested in vitro on U87MG-R cells and patient-derived Pt#3 TMZ-resistant cells, demonstrating the therapeutic efficacy based on cell viability assays. In vivo testing in the TMZ-resistant GBM orthotopic mouse model also showed promising anti-tumor activity, with treated mice demonstrating increased survival times compared to controls. In conclusion, our findings suggest that this drug targeting both CYP17A1 and HDAC6 presents a potential breakthrough in GBM-targeted therapy, addressing the pervasive issue of TMZ resistance.

Freeze-dried powder of daylily bud improves bromocriptine-induced lactation disorder in rats via JAK2/STAT5 pathway

Ethnopharmacological relevanceMilk deficiency is a prevalent problem in the world. Daylily (Hemerocallis citrina Borani), called the Chinese mother flower, is a traditional vegetable and is believed to possess a galactagogue effect in China. Flavonoids and phenols are considered as the active ingredients of daylily to promote lactation and improve depression. Aim of the studyThe aim of this study was to investigate the prolactin effects of freeze-dried powder of flower buds of H. citrina Baroni in rat and its action mechanisms. Materials and methodsThe chemical constituents of flower buds of H. citrina Baroni treated by different drying techniques were analyzed by ultrahigh pressure liquid chromatography-mass spectrometry. Sprague-Dawley (SD) rat model induced by bromocriptine was used to evaluate the effect of freeze-dried powder of daylily buds on promoting lactation. Network pharmacology method, ELISA, qPCR, and Western blot were used to clarify the action mechanisms. ResultsWe detected 657 compounds in daylily buds. The relative contents of total flavonoids and phenols in freeze-dried samples were higher than those in dried ones. Bromocriptine, as a dopamine receptor agonist, can significantly inhibit prolactin in rats. Daylily buds can restore the levels of prolactin, progesterone and estradiol depressed by bromocriptine, effectively improve the milk production of the rat, and promote the repair of rat mammary gland tissue. We analyzed the relationship between the chemical components of daylily buds and the genes related to lactation with network pharmacology method, revealing that flavonoids and phenols may be the active components that promoted milk production via JAK2/STAT5 pathway, which was confirmed by the results of qPCR and Western blot. Daylily buds can increase the mRNA expression of PRLR, CSN2, LALBA and FASN and the protein expression of PRLR, JAK2 and STAT5. ConclusionDaylily buds can improve the insufficient lactation of rats induced by bromocriptine through PRLR/JAK2/STAT5 pathway, and the freeze-dried processing method may better retain the active components of flavonoids and phenols that promote milk in daylily.

Identification and characterization of lignin depolymerization enzymes in Bacillus subtilis strain S11Y isolated from a tropical environment in Malaysia.

Biological pretreatment using microbial enzymes appears to be the most promising pre-treatment technology for the breakdown of recalcitrant lignin structure. This research focuses on the identification and characterization of lignin-depolymerizing enzymes in Bacillus subtilis strain S11Y, previously isolated from palm oil wastes in Malaysia. The draft genome sequences of this highly lignin-depolymerizing strain revealed that the genome lacked any of the well-known dye-decolorizing peroxidase or catalase-peroxidase that are commonly reported to be involved in lignin depolymerization by bacteria, indicating that strain S11Y has distinct sets of potential lignin depolymerization genes. The oxidative stress-related enzymes Cu/Zn type-superoxide dismutase (Sod2) and a heme-containing monofunctional catalase (Kat2) were identified in the genome sequences that are of interest. Their lignin-depolymerizing ability were evaluated by treating Alkali lignin (AL) with each enzyme and their degradation ability were evaluated using gel-permeation chromatography (GPC), ultrahigh-pressure liquid chromatography-mass spectrometry (UHPLC/MS), and gas chromatography-mass spectrometry (GC/MS), which successfully proved lignin depolymerizing ability. Successful evaluation of lignin depolymerizing enzymes can be applicable for lignin pretreatment process in green energy production and generation of valuable chemicals in bio-refinery.

Improved Performance of UHPLC–MS Hyphenated Systems

An ultrahigh-pressure liquid chromatography–mass spectrometry (UHPLC–MS) research prototype instrument was built to improve the resolution power and the usability of conventional LC–MS hyphenated instruments for routine analyses in pharmaceutical applications. The improved characteristics of this UHPLC–MS system include: 1) the dramatic reduction of post-column sample dispersion; 2) the adoption of vacuum jacketed columns (VJC) for the reduction of undesirable radial temperature gradients across the column diameter; and 3) the presence of a column outlet end nut heater to refocus the distorted peaks prior to analyte ionization. The benefits of each of these added features are analyzed with a rigorous approach from a peak broadening perspective. A 2x improvement in peak capacities recorded with this prototype UHPLC–MS system compared to a standard system (Acquity UHPLC I-class/Xevo TQ-S) is illustrated for the gradient separation of seven small pharmaceutical compounds using a 2.1 mm x 100 mm column packed with sub-2-μm core-shell particles (1.6 μm Acquity UHPLC Cortecs C18 column).

Clinical pharmacokinetics of kavalactones after oral dosing of standardized kava extract in healthy volunteers

Ethnopharmacological relevancePiper methysticum G. Forst. (Piperaceae), commonly known as kava, has been used as a traditional beverage for centuries for its relaxing properties. Kavalactones are considered to be the major constituents responsible for kava's beneficial effects. Despite the extensive use of kava, clinical pharmacokinetic data is not available in the literature; therefore, the findings of this study will be critical for the dosage calculations for future clinical evaluation of kava. Aim of the studyThe aim of the current study is to examine the clinical pharmacokinetics of six major kavalactones following oral dosing of flavokavain A/B-free standardized kava extract capsules in healthy volunteers using two dosage regimens. Materials and methodsA sensitive, reliable, and specific ultra-high pressure liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantification of six major kavalactones (kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin) and two flavokavains (A and B) in human plasma. Pharmacokinetic profiles were assessed in ten healthy volunteers after oral doses of standardized kava product, and plasma samples were analyzed for six kavalactones and two flavokavains using the validated UPLC-MS/MS method. Concentration-time data was subjected to pharmacokinetic analysis. ResultsThe systemic exposure of the kavalactones was found to be in the following order: dihydrokavain > dihydromethysticin > kavain > methysticin > yangonin. Desmethoxyyangonin was quantifiable only at a couple of time points, while flavokavain A and flavokavain B were not present in any of the plasma samples. Fast absorption of five kavalactones was observed with time to reach the maximum plasma concentration of 1–3 h. A dose proportionality in pharmacokinetics was established from 75 to 225 mg of kavalactone doses. In the multiple-dose study, a significant reduction in the extent of absorption of kavalactones with food was observed. ConclusionSingle and multiple-dose clinical pharmacokinetic studies for kava were performed in healthy volunteers, and higher exposure to the kavalactones was observed after single-dosing (225 mg), while a longer duration of exposure was observed after three times a day (3 x 75 mg) dosing.

CYP2C19 Plays a Major Role in the Hepatic N-Oxidation of Cotinine.

The primary mode of metabolism of nicotine is via the formation of cotinine by the enzyme CYP2A6. Cotinine undergoes further CYP2A6-mediated metabolism by hydroxylation to 3-hydroxycotinine and norcotinine, but can also form cotinine-N-glucuronide and cotinine-N-oxide (COX). The goal of this study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway. Specific inhibitors of major hepatic cytochrome P450 (P450) enzymes were used in cotinine-N-oxidation reactions using pooled human liver microsomes (HLMs). COX formation was monitored by ultrahigh-pressure liquid chromatography-tandem mass spectrometry and enzyme kinetic analysis was performed using microsomes from P450-overexpressing human embryonic kidney 293 (HEK293) cell lines. Genotype-phenotype analysis was performed in a panel of 113 human liver specimens. Inhibition of COX formation was only observed in HLMs when using inhibitors of CYP2A6, CYP2B6, CYP2C19, CYP2E1, and CYP3A4. Microsomes from cells overexpressing CYP2A6 or CYP2C19 exhibited similar N-oxidation activity against cotinine, with maximum reaction rate over Michaelis constant values (intrinsic clearance) of 4.4 and 4.2 nL/min/mg, respectively. CYP2B6-, CYP2E1-, and CYP3A4-overexpressing microsomes were also active in COX formation. Significant associations (P < 0.05) were observed between COX formation and genetic variants in CYP2C19 (*2 and *17 alleles) in HLMs. These results demonstrate that genetic variants in CYP2C19 are associated with decreased COX formation, potentially affecting the relative levels of cotinine in the plasma or urine of smokers and ultimately affecting recommended smoking cessation therapies. SIGNIFICANCE STATEMENT: This study is the first to elucidate the enzymes responsible for cotinine-N-oxide formation and genetic variants that affect this biological pathway. Genetic variants in CYP2C19 have the potential to modify nicotine metabolic ratio in smokers and could affect pharmacotherapeutic decisions for smoking cessation treatments.

Application of solvent pH under pressurized conditions using accelerated solvent extraction and green solvents to extract phytonutrients from wild berries

Berry fruits are known as “superfoods” due to health benefits associated with the high content of bioactive compounds or phytonutrients. Organic extraction solvents are fundamentally volatile compounds acquired from non-renewable resources, i.e petroleum-based solvents are presumed to be harmful to both human health and the environment. Owing to the new emphasis on environmental and safety protections and the development of green chemistry, finding alternative solvents to replace the organic ones has recently become a major concern. Indeed, there is a great interest in the scientific community to develop a suitable green approach to allow the extraction and recovery of the bioactive phytonutrients from berry sources for applications in improving human health and nutrition. In this study, the phytonutrient composition of three popular wild berries: blueberry (Vaccinium angustifolium), partridgeberry (Vaccinium vitis-idaea L.) and Rosehipberry (Rosa canina), were evaluated following extraction using biphasic green solvents at pH 2.5, 7.0, and 11.5 under pressurized conditions using Accelerated Solvent Extraction (ASE) system. The total phenolic content and composition, antioxidant activity, vitamin and mineral content of the berry extracts were evaluated using Folin-Ciocalteu assay, Ferric Reducing Antioxidant Power Assay (FRAP), Ultra-High-pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively. The highest total antioxidant activity (p ≤ 0.05) and phenolic content were observed in rosehip berry followed by blueberry and partridgeberry in the hydroethanolic extracts at pH 11.5. Nevertheless, the highest extraction efficiency of total fat-soluble vitamins (FSV) and total B vitamins were also observed in the hydroethanolic extract at pH 11.5. Both in rosehipberry and partridgeberry the highest vitamin C content was observed in hydroethanolic extract at pH 11.5, whereas in blueberry it was observed in the pH 2.5 hydroethanolic extract. In the present study, results demonstrated that using the green solvent system (hydroethanolic, 70:30 v/v) at alkaline pH (11.5) as a pressurized fluid in the ASE system provided significant recovery of phytonutrients from wild berries. This green environmentally friendly system of extraction could represent a suitable alternative for the preparation of food grade or green solvent-based extracts for applications in the food and nutraceutical industry.

Dark Iron-Catalyzed Reactions in Acidic and Viscous Aerosol Systems Efficiently Form Secondary Brown Carbon.

Iron-driven secondary brown carbon formation reactions from water-soluble organics in cloud droplets and aerosols create insoluble and soluble products of emerging atmospheric importance. This work shows, for the first time, results on dark iron-catalyzed polymerization of catechol forming insoluble black polycatechol particles and colored water-soluble oligomers under conditions characteristic of viscous multicomponent aerosol systems with relatively high ionic strength (I = 1-12 m) and acidic pH (∼2). These systems contain ammonium sulfate (AS)/nitrate (AN) and C3-C5 dicarboxylic acids, namely, malonic, malic, succinic, and glutaric acids. Using dynamic light scattering (DLS) and ultra high pressure liquid chromatography-mass spectrometry (UHPLC-MS), we show results on the rate of particle growth/agglomeration and identity of soluble oligomeric reaction products. We found that increasing I above 1 m and adding diacids with oxygen-to-carbon molar ratio (O:C > 1) significantly reduced the rate of polycatechol formation/aggregation by a factor of 1.3 ± 0.4 in AS solution in the first 60 min of reaction time. Using AN, rates were too slow to be quantified using DLS, but particles formed after 24 h reaction time. These results were explained by the relative concentration and affinity of ligands to Fe(III). We also report detectable amounts of soluble and colored oligomers in reactions with a slow rate of polycatechol formation, including organonitrogen compounds. These results highlight that brown carbon formation from iron chemistry is efficient under a wide range of aerosol physical states and chemical composition.

Capillary ultrahigh-pressure liquid chromatography-mass spectrometry for fast and high resolution metabolomics separations

LC-MS is an important tool for metabolomics due its high sensitivity and broad metabolite coverage. The goal of improving resolution and decreasing analysis time in HPLC has led to the use of 5 – 15 cm long columns packed with 1.7 – 1.9 µm particles requiring pressures of 8 – 12 kpsi. We report on the potential for capillary LC-MS based metabolomics utilizing porous C18 particles down to 1.1 µm diameter and columns up to 50 cm long with an operating pressure of 35 kpsi. Our experiments show that it is possible to pack columns with 1.1 µm porous particles to provide predicted improvements in separation time and efficiency. Using kinetic plots to guide the choice of column length and particle size, we packed 50 cm long columns with 1.7 µm particles and 20 cm long columns with 1.1 µm particles, which should produce equivalent performance in shorter times. Columns were tested by performing isocratic and gradient LC-MS analyses of small molecule metabolites and extracts from plasma. These columns provided approximately 100,000 theoretical plates for metabolite standards and peak capacities over 500 in 100 min for a complex plasma extract with robust interfacing to MS. To generate a given peak capacity, the 1.1 µm particles in 20 cm columns required roughly 75% of the time as 1.7 µm particles in 50 cm columns with both operated at 35 kpsi. The 1.1 µm particle packed columns generated a given peak capacity nearly 3 times faster than 1.7 µm particles in 15 cm columns operated at ~10 kpsi. This latter condition represents commercial state of the art for capillary LC. To consider practical benefits for metabolomics, the effect of different LC-MS variables on mass spectral feature detection was evaluated. Lower flow rates (down to 700 nL/min) and larger injection volumes (up to 1 µL) increased the features detected with modest loss in separation performance. The results demonstrate the potential for fast and high resolution separations for metabolomics using 1.1 µm particles operated at 35 kpsi for capillary LC-MS.

A Lipidome Map of the Silkworm Bombyx mori: Influences of Viral Infection.

Lipids have been recently proposed as key molecules for virus entry and egress, and lipid biosynthesis and signaling were reported necessary for some viruses during replication and infection. The silkworm Bombyx mori is an important economic insect and a model organism, but its lipid profiles have not been systematically investigated. Most silkworm strains are susceptible to the B. mori nuclear polyhedrovirus (BmNPV), a baculovirus that causes serious loss to the sericulture industry. Previously, our lab has screened a natural mutant of B. mori that is highly resistant to BmNPV. In this study, a comprehensive lipidomic analysis by ultrahigh pressure liquid chromatography-mass spectrometry (UPLC-MS) was carried out on the BmNPV-susceptible strain 306 and resistant strain NB (data deposited in MetaboLight MTBLS2142). Comparisons of the lipid profiles between the two strains reveal that phosphosphingolipids, diacylglycerolipids, ceramides, and quinones were present at notably higher levels in the susceptible strain, while lysophosphocholines were found at a higher level in the resistant strain. BmNPV administration changed the lipid profiles in both strains, revealing key lipids involved in virus infection and immune response. Some key enzymes in the lipid biosynthesis pathway were analyzed for their activities in the two silkworm strains and their virus-administered counterparts, underlining the relation among lipid biosynthesis, viral resistance, and immune response in the host.

Abstract P3-02-04: Combined aurora kinase A and mTORC 1/2 inhibition results in disruption of tumor cell energetics in triple negative breast cancer

Abstract Background: We have previously demonstrated the efficacy of pharmacologic targeting of Aurora Kinase A (AurA) in cell line and patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC). We observed that inherent or acquired resistance to AurA inhibitors was associated with a senescence phenotype and up-regulation of the PI3K/Akt/mTOR pathway. This provided the rationale to assess whether this resistance could be overcome with the combination of the AurA inhibitor alisertib with the TORC1/2 inhibitor TAK-228. In select TNBC PDX models, we found that there were highly significant anti-tumor effects of the combination compared to single agents. The purpose of the current study was to evaluate the potential role for alterations in tumor metabolic profiles in anti-tumor activity after dual inhibition of AurA and TORC1/2, as both pathways are known to mediate cellular metabolism and energy utilization in normal and cancer cells. Methods: Three TNBC PDX models were used for these studies. Tumor sections were implanted subcutaneously on bilateral flanks of athymic nude mice. When tumors reached ~150 mm3 mice were randomized into four treatment arms: vehicle; alisertib (30 mg/kg); TAK-228 (0.75 mg/kg); or the combination and dosed daily by oral gavage for 30 days (n=10 tumors per arm). At end of study, tumors were harvested and polar metabolites extracted for global ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS)-based metabolomics with a focus on central carbon and nitrogen metabolism. Metabolites were separated using a high throughput gradient-based C18 method and identified and validated manually against an in-house library of 800+ compounds. Results: Global metabolomics analysis of PDX tumors revealed a significant impact of TAK-228 treatment on fatty acid oxidation, resulting in increased levels of saturated fatty acids (C8, C10, C12) and palmitoleic acid, an increase in arginine, proline, metabolism and a decrease in glutamine metabolism. Alisertib treatment impacted tryptophan metabolism; initial results suggest a disruption of metabolite balance downstream of tryptophan. These disruptions in nitrogen and carbon metabolism occurred in the combination treatment arm as well. Of note, glucose utilization via glycolysis and the pentose phosphate pathway (PPP) were decreased with treatments, with the most profound decrease in the combination arm. Conclusions: In addition to increased anti-tumor growth activity, TNBC PDX tumor metabolic profiles were altered in mice treated alisertib, TAK-228 and the combination. Global metabolomics analysis revealed that several key metabolic pathways, including fatty acid oxidation, amino acid metabolism, glycolysis and PPP activity were significantly dysregulated, likely contributing to the observed tumor growth inhibition. Importantly, the effects on several pathways by the single agents persisted in the combination, while others, including the decrease in glycolysis and PPP activity, were more pronounced in the combination arm. These results support the hypothesis that dual targeting of AurA and TORC1/2 may exert potent anti-tumor activity in TNBC through several mechanisms, including the disruption of tumor cell energetics. This combination is currently being evaluated in a Phase I clinical trial with planned expansion cohorts with serial tumor biopsies (NCT02719691). These clinical samples may provide a means for validation of these preclinical observations and allow continued elucidation of the potential antitumor metabolic reprogramming resulting from this dual targeting strategy and the clinical setting most likely to benefit from this combination. Citation Format: John J Tentler, Angelo D'Allesandro, Julie A Reisz, Kyrie L Dailey, Anna Capasso, Anastasia Ionkina, Stacey M Bagby, Todd M Pitts, Jennifer R Diamond. Combined aurora kinase A and mTORC 1/2 inhibition results in disruption of tumor cell energetics in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-04.

Serum FFAs profile analysis of Normal weight and obesity individuals of Han and Uygur nationalities in China

BackgroundHan and Uygur are the two main nationalities living in Xinjiang, China. There are significant differences in the incidence of metabolic diseases for two nationalities, but the specific reasons are not clear. Obesity is an important risk factor for the development of metabolic syndrome, which may be closely related to the increase of serum free fatty acids (FFAs) content. This study aims to use metabolomics to compare the changes of serum FFAs profiles between normal weight (NW) and obese (OB) individuals of two nationalities, screening out the differential FFAs, predicting and evaluating their relationship with diseases.MethodsThirty-four kinds of FFAs in serum were detected by ultra-high-pressure liquid chromatography–mass spectrometry (UHPLC-MS) and distinctions in FFAs profiles were evaluated using a metabolomics method while Receiver operating characteristics (ROC) and logistic regression models were used to explore FFAs significant for diagnosing obesity and obesity-associated comorbidities.ResultsIn the Han nationality, ten kinds of FFAs (C7:0, C8:0, C9:0, C10:0, C11:0, C14:0, C18:2, C20:3, C20:4 and C22:6) showed significant differences between NW and OB individuals. These differential FFAs may be related to hypertension and gestational diabetes mellitus. In the Uygur nationality, C20:3 and C20:5 showed significant differences between NW and OB individuals. C9:0 and C19:0, which were screened out among the female subjects, showed a good ability to predict obesity status in Uygur females (AUC = 0.950).ConclusionIn both the Han and Uygur nationalities, the FFAs profiles of NW individuals differed from those of OB individuals. The significantly differential FFAs are closely related to obesity and may be important risk factors for obesity and related metabolic diseases.

Imaging Mass Spectrometry–Based Lipidomic Approach to Classification of Architectural Features in Nevi

Imaging Mass Spectrometry–Based Lipidomic Approach to Classification of Architectural Features in Nevi

3300 Progesterone receptor alters lipid biology in luminal breast cancer

OBJECTIVES/SPECIFIC AIMS: These studies seek to evaluate hormonal regulation of luminal breast cancer lipid metabolism and to identify targetable progesterone-mediated changes in lipid biology that contribute to therapeutic resistance in breast cancer. METHODS/STUDY POPULATION: Established and patient-derived luminal breast cancer cell lines, which express ER and PR, were used for this study. RNA transcript and protein expression levels were evaluated by qRT-PCR and immunoblot, respectively. Broad scale lipidomics of progesterone-treated cells was conducted via ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) through the UCD Skaggs School of Pharmacy Mass Spectrometry Core. RESULTS/ANTICIPATED RESULTS: Data mining of previously published microarray data of CK5+ and CK5− syngeneic cancer sublines revealed that CK5+ cells have increased expression of lipid processing genes, including LPL and PPARG. As progestin treatment induces a subpopulation of cells to turn on CK5 expression in luminal breast cancers, UHPLC-MS-based lipidomics analysis will expose whether modulation of the lipid landscape occurs in all cells with progesterone treatment, or whether this phenomenon is heightened specifically in CK5+ cells. I also expect that ER+ breast cancers with progestin induced-altered lipid content, such as lipid droplet formation, will evade therapy-induced death. DISCUSSION/SIGNIFICANCE OF IMPACT: There are numerous approved and developmental therapeutics targeting lipid biology. By determining if progestins alter lipid metabolic genes specifically in CK5+ CSCs, which are endocrine resistant, strategies may be devised to target these resistant cells using combination therapy in conjunction with existing therapies to prevent tumor recurrence.

Expired Epinephrine Maintains Chemical Concentration and Sterility

Objectives: Epinephrine shortages affect nearly all American emergency medical services (EMS) systems. Utilization of expired epinephrine could mitigate this situation in daily EMS operations. Concerns about using expired medications include sterility, potency, and potential harmful chemical decay byproducts. There are no cross-platform analyses of sterility and chemical purity of multiple samples of expired parenteral epinephrine. We hypothesized that epinephrine injections will remain sterile and will retain their active ingredient's content for more than 30 months past expiration. Methods: Six parenteral epinephrine prefilled syringes, 1 mg/10 mL, with an expiration date of January 1, 2012 had been stored in the climate controlled setting of a hospital inpatient pharmacy where they remained until they were taken for chemical or microbial analysis 30 months after expiration. An unexpired parenteral epinephrine prefilled syringe content was used as a control. Contents of three separate syringes with expired content from the same lot and one control underwent ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) and nuclear magnetic resonance (NMR) to determine epinephrine content and stability. In parallel, contents of another three expired epinephrine syringes were analyzed for sterility by plating on aerobic, anaerobic, and fungal media in a hospital microbiology laboratory. The aerobic plates were checked for growth in 3 days, the anaerobic in 5 days, and the fungal in 28 days. Results: UHPLC-MS and NMR showed that content of epinephrine present in the original sample remained unchanged compared to the control. There was no statistical difference in the UHPLC-MS and NMR signal amplitudes between the control and the expired samples. No chemical degradation byproducts were detected using NMR. There was no growth of any bacteria or fungus. Conclusion: Recurrent epinephrine shortages impact EMS and hospital operations in the United States. Individual administrators may be hesitant to authorize use of expired pharmaceuticals due to perceived potential complications or fear of litigation. This study shows that the original parenteral epinephrine remains sterile and detectably pure more than 2.5 years after expiration. Further study of the sterility and chemical integrity of expired medications that had been subjected to the conditions of EMS vehicles may be a future research endeavor based on the aforementioned paradigm.

Red blood cells in hemorrhagic shock: a critical role for glutaminolysis in fueling alanine transamination in rats.

Red blood cells (RBCs) are the most abundant host cell in the human body and play a critical role in oxygen transport and systemic metabolic homeostasis. Hypoxic metabolic reprogramming of RBCs in response to high-altitude hypoxia or anaerobic storage in the blood bank has been extensively described. However, little is known about the RBC metabolism following hemorrhagic shock (HS), the most common preventable cause of death in trauma, the global leading cause of total life-years lost. Metabolomics analyses were performed through ultra-high pressure liquid chromatography-mass spectrometry on RBCs from Sprague-Dawley rats undergoing HS (mean arterial pressure [MAP], <30 mm Hg) in comparison with sham rats (MAP, >80 mm Hg). Steady-state measurements were accompanied by metabolic flux analysis upon tracing of in vivo-injected 13C15N-glutamine or inhibition of glutaminolysis using the anticancer drug CB-839. RBC metabolic phenotypes recapitulated the systemic metabolic reprogramming observed in plasma from the same rodent model. Results indicate that shock RBCs rely on glutamine to fuel glutathione (GSH) synthesis and pyruvate transamination, whereas abrogation of glutaminolysis conferred early mortality and exacerbated lactic acidosis and systemic accumulation of succinate, a predictor of mortality in the military and civilian critically ill populations. Glutamine is here identified as an essential amine group donor in HS RBCs, plasma, liver, and lungs, providing additional rationale for the central role glutaminolysis plays in metabolic reprogramming and survival following severe hemorrhage.