High-resolution Liquid Chromatography-tandem Mass Spectrometry Research Articles

Different Proteins as Biomarkers for Sac Shrinkage After Endovascular Aortic Repair of Abdominal Aortic Aneurysms.

This study aims to identify circulating biomarkers by using proteomic analysis associated with sac shrinkage or expansion in patients undergoing endovascular aneurysm repair (EVAR) for abdominal aortic aneurysms (AAAs). Plasma samples were analysed from 32 patients treated with EVAR between 10/2009 and 10/2020. Patients were divided into two groups based on postoperative sac behaviour: sac shrinkage (≥5 mm reduction) and no shrinkage (stabilisation or expansion). Proteomic analysis was performed using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), with abundant protein depletion to enhance the detection of low-abundant proteins. Of the 32 patients, 20 exhibited sac shrinkage, and 12 showed no shrinkage. Proteomic analysis identified 632 proteins, with significant differential abundance observed after adjusting for relevant clinical parameters. Notably, neurogranin (NRGN) levels were significantly associated with hypertension and smoking, while casein alpha S1 (CSN1S1) levels varied with statin use. Differentially abundant proteins related to aortic diameter included calpastatin, SCUBE3, and ubiquitin-conjugating enzyme E2, among others. Proteomic profiling revealed distinct biomarker patterns associated with sac behaviour in EVAR-treated AAA patients. These findings suggest potential therapeutic targets for enhancing EVAR outcomes and underscore the need for further investigation into the biological mechanisms underlying aneurysm sac shrinkage and stability.

The biosynthesis and impacts of cytokinins on growth of the oyster mushroom, Pleurotus ostreatus

ABSTRACT While a lot is known about cytokinins (CKs) and their actions at the molecular and cellular levels in plants, much less is known about the function of CKs in other kingdoms such as fungi. CKs have been detected in a wide range of fungal species where they play roles ranging from enhancing the virulence of phytopathogens to fortifying plant growth when secreted from fungal symbionts. However, the role of CKs where they concern fungal physiology, apart from plant associations, remains largely uncharacterized. Profiling by UHPLC-HRMS/MS (ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry) revealed that Pleurotus ostreatus (oyster mushroom) produces CKs in vitro in both liquid and solid cultures. During fungal growth, CK profiling patterns were consistent with previous suggestions that tRNA degradation products might play a role in the physiological development of fungi. It confirms that those products are CKs that act as fungal growth regulators. Moreover, P. ostreatus was shown to respond to exogenous applications of aromatic and isoprenoid CKs, and their effects were dependent on the dose and CK type in a biphasic manner consistent with hormone action. N 6-benzyladenine (BAP), kinetin (KIN), N 6-isopentenyladenine (iP), and trans-zeatin (tZ) bioassays all revealed hormesis-type responses. Accordingly, at low doses, mycelium colony diameter, biomass accumulation, and changes in morphology were stimulated, whereas at high doses only inhibitory effects were observed. Thus, CKs may act as “mycohormones” and consequently have potential for applications in fungal agriculture and medicinal compound production.

Cost-effective synthesis and application of Cu²⁺-doped melamine formaldehyde resin for enhanced tetracycline enrichment in environmental water

Tetracycline antibiotics (TCs) are typically present at low residue levels in environmental water, necessitating enrichment prior to analysis. In this study, a Cu2+-doped melamine formaldehyde resin (Cu-MFR) was synthesized to enhance the adsorption efficiency for TCs, leveraging the formation of stable Cu2+-TC complexes on the sorbent surface. Then it was used as an adsorbent in solid-phase extraction (SPE) for the enrichment of four TCs from water samples. The optimized parameters for Cu-MFR in the analysis of TCs in environmental water enabled linear detection ranges from 0.20 to 50 ng/mL, with relative recoveries ranging from 79.5 % to 97.6 % and relative standard deviations ≤ 10.0 %, using high-performance liquid chromatography-tandem high-resolution mass spectrometry (HPLC-HRMS). The limits of detection for the four TCs were determined to be between 0.11 and 0.16 ng/mL, indicating its potential for practical application in real sample analysis.

Consolidating two-dimensional liquid chromatography–high-resolution tandem mass spectrometry (LC×LC-HRMS/MS) technique for the non-targeted analysis of poly- and perfluorinated substances: A trial on aqueous film-forming foams

To date, poly- and perfluoroalkyl substances (PFAS) represent a real threat for their environmental persistence, wide physicochemical variability, and their potential toxicity. Thus far a large portion of these chemicals remain structurally unknown. These chemicals, therefore, require the implementation of complex non-targeted analysis workflows using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for their comprehensive detection and monitoring. This approach, even though comprehensive, does not always provide the much-needed analytical resolution for the analysis of complex PFAS mixtures such as fire-fighting aqueous film-forming foams (AFFFs). This study consolidates the advantages of the LC×LC technique hyphenated with high-resolution tandem mass spectrometry (HRMS/MS) for the identification of PFAS in AFFF mixtures. A total of 57 PFAS homolog series (HS) were identified in 3M and Orchidee AFFF mixtures thanks to the (i) high chromatographic peak capacity (n’2D,c ~ 300) and the (i) increased mass domain resolution provided by the “remainder of Kendrick Mass” (RKM) analysis on the HRMS data. Then, we attempted to annotate the PFAS of each HS by exploiting the available reference standards and the FluoroMatch workflow in combination with the RKM defect by different fluorine repeating units, such as CF2, CF2O, and C2F4O. This approach resulted in 12 identified PFAS HS, including compounds belonging to the HS of perfluoroalkyl carboxylic acids (PFACAs), perfluoroalkyl sulfonic acids (PFASAs), (N-pentafluoro(5)sulfide)-perfluoroalkane sulfonates (SF5-PFASAs), N-sulfopropyldimethylammoniopropyl perfluoroalkane sulfonamides (N-SPAmP-FASA), and N-carboxymethyldimethylammoniopropyl perfluoroalkane sulfonamide (N-CMAmP-FASA). The annotated categories of perfluoroalkyl aldehydes and chlorinated PFASAs represent the first record of PFAS HS in the investigated AFFF samples.

Structural elucidation of tramadol, its derivatives, and metabolites using chemical derivatization and liquid chromatography-high-resolution tandem mass spectrometry.

Tramadol (T) is a strong painkiller drug that belongs to the opioid analgesic group. Several accidental intoxication cases after oral administration of T have been reported in the past decade. Tramadol, its derivatives, and metabolites present information-limited mass spectra with one prominent peak representing the amine-containing residue; therefore, their structural determination based on both electron impact mass spectrometry (EI-MS) and ESI-MS/MS spectra could be misleading. A novel analytical method for the structural elucidation of tramadol, its four homologs, and its two main phase I metabolites (N-desmethyltramadol and O-desmethyltramadol) was developed using chemical modification and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS) with Orbitrap technology. After chemical derivatization, each of the investigated T series exhibited informative mass spectra that enabled better exposition of their structures. The developed method was successfully implemented to explicitly identify the structures of tramadol and its N-desmethyltramadol metabolite in urine samples at low ng/mL levels. An efficient derivatization-aided strategy was developed for rapidly elucidating the structure of tramadol-like compounds. The method is intended to assist forensic chemists in better diagnosing T and its analogs and metabolites in clinical or forensic toxicology laboratories.

In Vitro and In Vivo Human Metabolism of Ostarine, a Selective Androgen Receptor Modulator and Doping Agent.

Ostarine (enobasarm) is a selective androgen receptor modulator with great therapeutic potential. However, it is also used by athletes to promote muscle growth and enhance performances without the typical adverse effects of anabolic steroids. Ostarine popularity increased in recent years, and it is currently the most abused "other anabolic agent" (subclass S1.2. of the "anabolic agents" class S1) from the World Anti-Doping Agency's (WADA) prohibited list. Several cases of liver toxicity were recently reported in regular users. Detecting ostarine or markers of intake in biological matrices is essential to document ostarine use in doping. Therefore, we sought to investigate ostarine metabolism to identify optimal markers of consumption. The substance was incubated with human hepatocytes, and urine samples from six ostarine-positive cases were screened. Analyses were performed via liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) and software-assisted data mining, with in silico metabolite predictions. Ten metabolites were identified with hydroxylation, ether cleavage, dealkylation, O-glucuronidation, and/or sulfation. The production of cyanophenol-sulfate might participate in the mechanism of ostarine liver toxicity. We suggest ostarine-glucuronide (C25H22O9N3F3, diagnostic fragments at m/z 118, 185, and 269) and hydroxybenzonitrile-ostarine-glucuronide (C25H22O10N3F3, diagnostic fragments at m/z 134, 185, and 269) in non-hydrolyzed urine and ostarine and hydroxybenzonitrile-ostarine (C19H14O4N3F3, diagnostic fragments at m/z 134, 185, and 269) in hydrolyzed urine as markers to document ostarine intake in doping.

Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) β-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG β-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Mycotoxin contamination profiling in coffee bean by targeted LC-HRMS

Abstract Indonesia is the fourth coffee producer in the world which is 95% produced by smallholder plantations with various ways of processing and storage in the form of green beans and roasted beans. This study aims to analyse the presence of mycotoxin contamination of Menoreh coffee in various storage levels using Liquid Chromatography tandem High-Resolution Mass Spectrometry (LC-HRMS) analysis. The analysis of mycotoxin contamination in green beans and roasted beans is crucial to ensure the safety and quality of the coffee for consumption. This study used a random sampling method. The samples tested were Menoreh Robusta coffee bean from two processors in Samigaluh, Kulon Progo, Yogyakarta, Indonesia. The results showed that green beans stored in the dryer house for more than 6 months were contaminated with aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), ochratoxin, and patulin with 24.48; 10.98; 57.18 and 16.92 of area max (106), respectively. Samples of broken green beans stored in contaminated warehouses were contaminated with patulin. However, all roasted bean and roasted bean samples that have been mashed do not contain aflatoxin B1, B2, or patulin. Targeted metabolomic using LC-HRMS profiling is a powerful tool for the rapid detection of mycotoxin in coffee bean. This method could be developed for quantitative analysis to provide accurate concentration.

Development of an LC-HRMS/MS Method for Quantifying Steroids and Thyroid Hormones in Capillary Blood: A Potential Tool for Assessing Relative Energy Deficiency in Sport (RED-S).

Relative energy deficiency in sport (RED-S) is a condition that arises from persistent low energy availability (LEA), which affects the hypothalamic-pituitary axis and results in alterations of several hormones in both male and female athletes. As frequent blood hormone status determinations using venipuncture are rare in sports practice, microsampling offers promising possibilities for preventing and assessing RED-S. Therefore, this study aimed at developing a liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) method for quantifying relevant steroids and thyroid hormones in 30 μL of capillary blood obtained using Mitra® devices with volumetric absorptive microsampling technology (VAMS®). The results of the study showed that all validation criteria were met, including a storage stability of more than 28 days in a frozen state (-18 °C) and 14 days at room temperature (20 °C). The validated assay provided precise (<12%) and accurate (<13%) results for all the target analytes. Furthermore, as a proof of concept, autonomously collected VAMS® samples from 50 female and male, healthy, active adults were analyzed. The sensitivity of all analytes was adequate to quantify the decreased hormone concentrations in the RED-S state, as all authentic samples could be measured accordingly. These findings suggest that self-collected VAMS® samples offer a practical opportunity for regular hormone measurements in athletes and can be used for early RED-S assessment and progress monitoring during RED-S recovery.

A novel strategy for detoxification of deoxynivalenol via modification of both toxic groups

Deoxynivalenol (DON) is the most abundant mycotoxin in cereal crops and derived foods and is of great concern in agriculture. Bioremediation strategies have long been sought to minimize the impact of mycotoxin contamination, but few direct and effective enzyme-catalyzed detoxification methods are currently available. In this study, we established a multi-enzymatic cascade reaction and successfully achieved detoxification at double sites: glutathionylation for the C-12,13 epoxide group and epimerization for the C-3 hydroxyl group. This yielded novel derivatives of DON, 3-epi-DON-13-glutathione (3-epi-DON-13-GSH) as well as its by-product, 3-keto-DON-13-GSH, for which precise structures were validated via liquid chromatography–high-resolution tandem mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Both cell viability and DNA synthesis assays demonstrated dramatically decreased cytotoxicity of the double-site modified product 3-epi-DON-13-GSH. These findings provide a promising and urgently needed novel method for addressing the problem of DON contamination in agricultural and industrial settings.

Post-metabolism impurity profiling of carfentanil, remifentanil, sufentanil, and benzylfentanyl

Carfentanil, remifentanil, and sufentanil are potent fentanyl analogues that are regularly mixed with illicit drugs causing many overdose deaths. Chemical impurity profiling of these drugs is a well-established technique for linking evidence found at a crime scene to other seized samples. The current study aims to expand the application of impurity profiling to metabolized samples to find synthesis specific markers. This is particularly relevant when the drug has been consumed, and no intact material is present at a crime scene. Carfentanil, remifentanil, and sufentanil were synthesized according to the Ugi or 7-step method and benzylfentanyl was produced using the Siegfried method. After in-vitro metabolism with human liver microsomes, the samples were analyzed by gas chromatography-mass spectrometry (GC–MS) and liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS). Characteristic markers were found by applying a match criterion approach and principal component analysis (PCA). The precursors 4-ANBP, aniline, and N-phenylacetamide and several metabolites were identified in post-metabolism samples, indicating that specific synthesis information is retained after in-vitro metabolism. The detected levels were in line with concentrations reported in case work. In addition, LDA was applied to maximize discrimination between synthesis methods and to establish likelihood ratios (LRs). Calibrated LR values were in the range of 0.083 to 16 with very low false positive and false negative error rates. In conclusion, the presented work demonstrates the possibility of combining chemical profiling and retrospective biomarker analysis to obtain information about the synthesis method, which could be useful for forensic reconstructions and attribution investigations.

In Vivo and In Vitro Metabolic Fate and Urinary Detectability of Five Deschloroketamine Derivatives Studied by Means of Hyphenated Mass Spectrometry.

Ketamine derivatives such as deschloroketamine and deschloro-N-ethyl-ketamine show dissociative and psychoactive properties and their abuse as new psychoactive substances (NPSs) has been reported. Though some information is available on the biotransformation of dissociative NPSs, data on deschloro-N-cyclopropyl-ketamine deschloro-N-isopropyl-ketamine and deschloro-N-propyl-ketamine concerning their biotransformation and, thus, urinary detectability are not available. The aims of the presented work were to study the in vivo phase I and II metabolism; in vitro phase I metabolism, using pooled human liver microsomes (pHLMs); and detectability, within a standard urine screening approach (SUSA), of five deschloroketamine derivatives. Metabolism studies were conducted by collecting urine samples from male Wistar rats over a period of 24 h after their administration at 2 mg/kg body weight. The samples were analyzed using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) and gas chromatography-mass spectrometry (GC-MS). The compounds were mainly metabolized by N-dealkylation, hydroxylation, multiple oxidations, and combinations of these metabolic reactions, as well as glucuronidation and N-acetylation. In total, 29 phase I and 10 phase II metabolites were detected. For the LC-HRMS/MS SUSA, compound-specific metabolites were identified, and suitable screening targets could be recommended and confirmed in pHLMs for all derivatives except for deschloro-N-cyclopropyl-ketamine. Using the GC-MS-based SUSA approach, only non-specific acetylated N-dealkylation metabolites could be detected.

Nontargeted Analysis of Coumarins in Source Water and Their Formation of Chlorinated Coumarins as DBPs in Drinking Water.

Coumarin was detected as one of the most abundant compounds by nontargeted analysis of natural product components in actual water samples prior to disinfection. More importantly, prechlorination of humic acid generated 3-hydroxycoumarin and monohydroxy-monomethyl-substituted coumarin with a total yield of ≤10.1%, which suggested the humic substance in raw water is an important source of coumarins. 7-Hydroxycoumarin, 6-hydroxy-4-methylcoumarin, 6,7-dihydroxycoumarin, and 7-methoxy-4-methylcoumarin were identified in raw water by high-performance liquid chromatography-tandem high-resolution mass spectrometry because only some coumarin standards were commercially available. Their chlorination generated monochlorinated and polychlorinated coumarins, and their structures were confirmed by the synthesized standards. These products could form at various dosages of chlorine and pH levels, and some with a concentration of 600 ng/L can be stable in tap water for days. 3,6,8-Trichloro-7-hydroxycoumarin, 3-chloro-7-methoxy-4-methylcoumarin, and 3,6-dichloro-7-methoxy-4-methylcoumarin were first identified in finished water with concentrations of 0.0670, 78.1, and 14.7 ng/L, respectively, but not in source water, suggesting that they are new DBPs formed during disinfection. The cytotoxicity of 3-chloro-7-methoxy-4-methylcoumarin in CHO-K1 cells was comparable to those of 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone in TIC-Tox analyses, suggesting that further investigation of their occurrence and control in drinking water systems is warranted.

Neuroretinal degeneration in a mouse model of systemic chronic immune activation observed by proteomics.

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.

Biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach using LC-HRMS/MS and chemometrics

Since its first employment in World War I, chlorine gas has often been used as chemical warfare agent. Unfortunately, after suspected release, it is difficult to prove the use of chlorine as a chemical weapon and unambiguous verification is still challenging. Furthermore, similar evidence can be found for exposure to chlorine gas and other, less harmful chlorinating agents. Therefore, the current study aims to use untargeted high resolution mass spectrometric analysis of chlorinated biomarkers together with machine learning techniques to be able to differentiate between exposure of plants to various chlorinating agents. Green spire (Euonymus japonicus), stinging nettle (Urtica dioica), and feathergrass (Stipa tenuifolia) were exposed to 1000 and 7500 ppm chlorine gas and household bleach, pool bleach, and concentrated sodium hypochlorite. After sample preparation and digestion, the samples were analyzed by liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). More than 150 chlorinated compounds including plant fatty acids, proteins, and DNA adducts were tentatively identified. Principal component analysis (PCA) and linear discriminant analysis (LDA) showed clear discrimination between chlorine gas and bleach exposure and grouping of the samples according to chlorine concentration and type of bleach. The identity of a set of novel biomarkers was confirmed using commercially available or synthetic reference standards. Chlorodopamine, dichlorodopamine, and trichlorodopamine were identified as specific markers for chlorine gas exposure. Fenclonine (Cl-Phe), 3-chlorotyrosine (Cl-Tyr), 3,5-dichlorotyrosine (di-Cl-Tyr), and 5-chlorocytosine (Cl-Cyt) were more abundantly present in plants after chlorine contact. In contrast, the DNA adduct 2-amino-6-chloropurine (Cl-Ade) was identified in both types of samples at a similar level. None of these chlorinated biomarkers were observed in untreated samples. The DNA adducts Cl-Cyt and Cl-Ade could clearly be identified even three months after the actual exposure. This study demonstrates the feasibility of forensic biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach.

Integrative leaf anatomy structure, physiology, and metabolome analyses revealed the response to drought stress in sainfoin at the seedling stage.

Sainfoin (Onobrychis viciaefolia) is a vital legume forage, and drought is the primary element impeding sainfoin growth. The anatomical structure, physiological indexes, and metabolites of the leaves of sainfoin seedlings with a drought-resistant line of P1 (DRL) and a drought-sensitive material of 2049 (DSM) were analyzed under drought (-1.0 MPa) with polyethylene glycol-6000 (PEG-6000). The leaf anatomy was studied by the paraffin section method. The related physiological indexes were measured by the hydroxylamine oxidation method, titanium sulfate colorimetric method, thiobarbituric acid method, acidic ninhydrin colorimetric method, and Coomassie brilliant blue method. The metabolomics analysis was composed of liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS). The results revealed that the thickness of the epidermis, palisade tissue, and sponge tissue of DRL were significantly greater than those of DSM. The leaves of DRL exhibited lower levels of superoxide anion (O2 •-) production rate, hydrogen peroxide (H2O2) content, and malondialdehyde (MDA) content compared with DSM, while proline (Pro) content and soluble protein (SP) content were significantly higher than those of DSM. A total of 391 differential metabolites were identified in two samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that the primary differential metabolites were concentrated into the tyrosine metabolism; isoquinoline alkaloid biosynthesis; ubiquinone and other terpenoid quinone biosynthesis; neomycin, kanamycin, and gentamicin biosynthesis; and anthocyanin biosynthesis metabolic pathways. Compared with DSM, DRL had more complete anatomical structure, lower active oxygen content, and higher antioxidant level. The results improved our insights into the drought-resistant mechanisms in sainfoin.

Abstract 1839: Proteomic and glycoproteomic differences associated with prostate cancer in urine

Abstract Background and Aims: Prostate cancer is one of the most common cancers affecting men in the United States, with ~288,300 new cases and 34,700 deaths in 2023 alone. Prostate-specific antigen (PSA), the most widely used prostate cancer biomarker, lacks specificity and sensitivity, leading to unnecessary biopsies, overdiagnosis, and overtreatment. There is an urgent unmet clinical need to find new biomarkers that can distinguish patients with benign disease from those with prostate cancer. Urine is an attractive source for these biomarkers as prostate-derived proteins are more highly concentrated in urine than in blood. To better understand both global proteomic and glycoproteomic molecular signatures that could lead to improved diagnostics for prostate cancer, we analyzed urine samples from unaffected men, men with benign prostatic hyperplasia (BPH), and men with prostate cancer. Methods: Urine samples (N = 58 total) were collected from unaffected men (N = 17), men with BPH (N = 24), and men with prostate cancer (N = 17). Samples were digested with trypsin and analyzed with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for shotgun proteomics analysis. Association with cancer was determined through supervised analysis using Pearson correlation. For glycoproteomics analysis, intact glycopeptides were enriched using strong anion exchange-electrostatic repulsion hydrophilic interaction chromatography, fractionated using high pH reversed-phase chromatography, and analyzed with LC-MS/MS. Association with the three conditions was determined through several rounds of supervised analysis using Pearson correlation. Results: In total, 3542 human proteins were identified, and 78 proteins (p &amp;lt; 0.01) were significantly associated with prostate cancer. The glycolysis pathway and lysosome pathway were enriched in samples from men with prostate cancer. In the glycoproteomics analysis, 36 proteins (p &amp;lt; 0.05) had altered glycan diversity in one of the three conditions, and the complement and coagulation cascade pathway and the lysosome pathway were highly enriched. Six proteins (p &amp;lt; 0.05) had significantly increased glycan diversity in the cancerous samples than in the BPH and normal samples. Conclusions: This study identifies a set of interesting proteomic and glycoproteomic differences in urine from men with prostate cancer compared to BPH and non-cancer samples, which warrant further investigation in additional sample cohorts as potential novel biomarkers. Citation Format: Nikhiya Shamsher, Fernando Garcia-Marques, Abel Bermudez, Mark R. Flory, Sharon J. Pitteri. Proteomic and glycoproteomic differences associated with prostate cancer in urine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1839.

In vitro, invivo metabolism and quantification of the novel synthetic opioid N-piperidinyl etonitazene (etonitazepipne).

N-piperidinyl etonitazene (etonitazepipne) is a newly synthesized opioid related to the 2-benzylbenzimidazole analog class. Etonitazepipne has been formally notified and placed under intensive monitoring in Europe in January 2022. Nitazenes have high affinity at µ-opioid receptor (MOR). Etonitazepipne, specifically shows a EC50 of 2.49 nM, suggesting about 50 times higher potency combined with higher efficacy compared to morphine. Antinociceptive potency l ('hot plate test' with rats) was 192-fold greater than that of morphine. Here we report on a post-mortem case involving etonitazepipne and its quantification using a standard addition method (SAM) through liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, characterization and identification of phase I human metabolites using invitro assay based on pooled human liver microsomes (pHLM) was performed along with the analysis of authentic urine samples by means of high-performance liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). The concentration of etonitazepipne in post-mortem blood and urine was 8.3 and 11 ng/mL, respectively. SAM was validated by assessing the following parameters: intraday and interday repeatability, matrix effect and recovery rate in post-mortem blood. A total of 20 and 14 metabolites were identified after pHLM incubation and urine analysis, respectively. Most pronounced invitro and invivo transformations were O-deethylation, hydroxylation, ketone reduction, and combinations thereof. Considering small traces of the parent drug often found in real cases, the identification of metabolic biomarkers is crucial to identify exposure to this drug. O-deethylated, oxidated metabolites, and combination thereof are proposed as urinary biomarkers along with the parent compound.

Occurrence of perfluoroalkyl substances in canned tuna and their impact on food safety

Continued use for industrial purposes of ever-new compounds compulsorily leads to the possibility of food contamination and risk for the consumer. Among the legacy and emerging contaminants, perfluoroalkyl substances (PFASs), used in the mid-20th century and still being implemented, are molecules of particular concern for their toxicity as endocrine disruptors, immunodepressants, possible carcinogens and liver and kidney toxicity. The main sources of PFAS exposure are water and food, particularly fish. In the European Union, European Food Safety Authority (EFSA) aimed the focus on 4 PFASs: perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexane sulfonic acid (PFHxS) and perfluorooctane sulfonic acid (PFOS). In this study, canned tuna was the investigated matrix, as a highly consumed food. The study aimed to detect the presence of 18 PFASs in 75 paired batches of precooked tuna loins and canned tuna through ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry (UHPLC-HRMS). Seven of the investigated PFASs were detected in the analyzed samples. Among them, just two of the four PFASs of which EFSA established a Tolerable Weekly Intake (TWI), i.e. PFOS and PFNA, were found in canned tuna. The risk characterization related to canned tuna resulted in an intake value much lower than the TWI for the average Italian consumer, but is a possible concern for high consumers, considering that canned tuna is not the only source of PFAS intake.

Targeted screening of the synergistic components in Artemisia annua L. leading to enhanced antiplasmodial potency of artemisinin based on a "top down" PD-PK approach

Ethnopharmacological relevanceArtemisinin (ART) showed enhanced antimalarial potency in the herb Artemisia annua L. (A. annua), from which ART is isolated. Increased absorption of ART with inhibited metabolism in the plant matrix is an underlying mechanism. Several synergistic components have been reported based on a "bottom-up" approach, i.e., traditional isolation followed by pharmacokinetic and/or pharmacodynamic evaluation. Aim of the studyIn this study, we employed a "top-down" approach based on in vivo antimalarial and pharmacokinetic studies to identify synergistic components in A. annua. Materials and methodsTwo A. annua extracts in different chemical composition were obtained by extraction using ethyl acetate (EA) and petroleum ether (PE). The synergistic antimalarial activity of ART in two extracts was compared both in vitro (Plasmodium falciparum) and in vivo (murine Plasmodium yoelii). For the PD-PK correlation analysis, the pharmacokinetic profiles of ART and its major metabolite (ART-M) were investigated in healthy rats after a single oral administration of pure ART (20 mg/kg) or equivalent ART in each A. annua extract. A liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS)-based analytical strategy was then applied for efficient component classification and structural characterization of the differential components in the targeted extract with a higher antimalarial potency. Major components isolated from the targeted extract were then evaluated for their synergistic effect in the same proportion. ResultsCompared with pure ART (ED50, 5.6 mg/kg), ART showed enhanced antimalarial potency in two extracts in vivo (ED50 of EA, 2.9 mg/kg; ED50 of PE, 1.6 mg/kg), but not in vitro (IC50, 15.0–20.0 nM). A significant increase (1.7-fold) in ART absorption (AUC0-t) was found in rats after a single oral dose of equivalent ART in PE but not in EA; however, no significant change in the metabolic capability (AUCART-M/AUCART) was found for ART in either extract. The differential component analysis of the two extracts showed a higher composition of sesquiterpene compounds, especially component AB (3.0% in PE vs. 0.9% in EA) and component AA (14.1% in PE vs. 5.1% in EA). Two target sesquiterpenes were isolated and identified as arteannuin B (AB) and artemisinic acid (AA). The synergism between ART and AB/AA in the same proportion with PE extract (20:1.6:7.6, mg/kg) was verified by a pharmacokinetic study in rats. ConclusionsA "top-down" strategy based on PD-PK studies was successfully employed to identify synergistic components for ART in A. annua. Two sesquiterpene compounds (arteannuin B and artemisinic acid) could enhance the antimalarial potency of ART by increasing its absorption.