Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors.

The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets.

Rapid determination of lipophilic vitamins in human serum by ultra-high performance liquid chromatography using a fluorinated column and high-throughput miniaturized liquid-liquid extraction.

A high-throughput miniaturized liquid-liquid extraction procedure followed by a simple ultra-high performance liquid chromatography method coupled with fluorescence detection for bioanalytical analysis of all tocopherol isomers and retinol in human serum has been developed and validated. In the extraction procedure, a synthetic internal standard tocol was used, which does not occur in the human body. The separation of structurally related vitamins was achieved using a new generation of pentafluorophenyl propyl core-shell stationary phase with elution using methanol and an aqueous solution of ammonium acetate. The fluorescence of retinol and tocopherol isomers was detected at λex  = 325, 295 nm and λem  = 480, 325 nm, respectively. The rapid baseline separation of all analytes was accomplished within 4.0 min. The sensitivity of method was demonstrated with lower limits of quantification: retinol 0.01 µM, α-tocopherol 0.38 µM, ß-tocopherol 0.18 µM, γ-tocopherol 0.14 µM, and δ-tocopherol 0.01 µM. Possible application of this method in clinical practice was confirmed by the analysis of human serum samples from healthy volunteers. Finally, the simultaneous determination of retinol and all tocopherol isomers in human serum can enable the clarification of their role in metabolism and in diseases such as cancer.

A fully validated bioanalytical method using an UHPLC-MS/MS system for quantification of DNA and RNA oxidative stress biomarkers.

A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.

Recent advances in the determination of tocopherols in biological fluids: from sample pretreatment and liquid chromatography to clinical studies.

Vitamin E comprises eight related compounds: α-, ß-, γ-, δ-tocopherols and α-, ß-, γ-, δ-tocotrienols. In the past, α-tocopherol has been the isomer that was studied most, and its anti-inflammatory and anti-proliferative effects have been described. Therefore, many prevention trials have investigated the effect of α-tocopherol on human health. Current research studies have also defined the important roles of other tocopherols, such as anti-inflammatory, anti-proliferative and cancer preventative effects. Knowledge of the individual tocopherols could help to understand their roles in various metabolic pathways. This review summarizes the recent trends in sample pretreatment, liquid chromatography and selected applications of the determination of tocopherols in various biological materials. The relationship between tocopherol isomers and serious diseases is also described. Graphical Abstract Article structure.

Prognostic Significance of Serum and Urinary Neopterin Concentrations in Patients with Rectal Carcinoma Treated with Chemoradiation.

AIM: To analyze the prognostic significance of serum and urinary neopterin concentrations in patients with rectal adenocarcinoma treated with (chemo)radiation. PATIENTS AND METHODS: Urinary and serum neopterin and peripheral blood cell count were determined in 49 patients with rectal carcinoma before the start of (chemo)radiation. RESULTS: Neopterin concentrations exhibited a significant inverse correlation with hemoglobin and positive correlation with leukocyte count, platelet count and platelet-to-lymphocyte ratio. Increased serum neopterin concentration was associated with significantly inferior relapse-free survival (RFS) and overall survival. However, a significant association was observed only in 28 patients treated in the neoadjuvant setting. Although increased urinary neopterin was also associated with inferior RFS and overall survival, this was not statistically significant. The neutrophil-to-lymphocyte ratio was also associated with poor prognosis. CONCLUSION: The data presented herein indicate a prognostic significance of serum neopterin concentrations in patients with rectal cancer treated with neoadjuvant chemoradiation.

Citrulline as a biomarker of gastrointestinal toxicity in patients with rectal carcinoma treated with chemoradiation.

BACKGROUND: Gastrointestinal toxicity is the principal toxicity of chemoradiation in the treatment of rectal carcinoma. The assessment of this toxicity still relies mostly on the symptoms reported by the patient. METHODS: Plasma citrulline, serum neopterin and urinary neopterin were followed weekly in 49 patients with rectal carcinoma during chemoradiation. RESULTS: Citrulline significantly (p<0.05) decreased while serum and urinary neopterin concentrations increased during therapy. Irradiated gut volume correlated significantly inversely with citrulline and positively with urinary neopterin. Statistically significant inverse correlations were also observed between urinary neopterin and plasma citrulline concentrations during the treatment. Urinary neopterin concentrations were significantly higher and citrulline concentrations were lower in patients who experienced grade ≥3 gastrointestinal toxicity. CONCLUSIONS: Citrulline represents a promising biomarker of gastrointestinal toxicity. Moreover, the volume of irradiated gut correlated with urinary neopterin concentrations and an association was observed between gastrointestinal toxicity evidenced by lower citrulline concentrations and systemic immune activation reflected in increased concentrations of urinary neopterin.

Fast and sensitive HPLC method for the determination of neopterin, kynurenine and tryptophan in amniotic fluid, malignant effusions and wound exudates.

AIM: A new HPLC method for the determination of neopterin, kynurenine and tryptophan using a second-generation monolith stationary phase and high-throughput sample preparation procedure based on microplates was developed and fully validated. MATERIALS & METHODS: As the stationary phase a monolithic C18 Chromolith high-resolution column with dimensions of 4.6 × 100 mm connected to a monolithic 4.6 × 10-mm security guard was used. Separation was achieved using 15 mM phosphate buffer (KH2PO4 +K2HPO4·3H2O at pH 3) and acetonitrile in gradient mode. RESULTS: Target analytes were determined in 5.5 minutes in amniotic fluid, effusions and wound exudates with a limit of quantification (LOQ) of 1.25 nM for neopterin, 2.5 µM for tryptophan and 0.25 µM for kynurenine. DISCUSSION: The method was applied to real clinical sample measurements, and it will be used to monitor neopterin, kynurenine and tryptophan levels in biological fluids to assess the patient response to therapy and clinical status.

Blood levels of antioxidants during age-related macular degeneration treatment by rheohaemapheresis.

AIMS: Rheohaemapheresis treatment influences rheological markers and most likely improves metabolism in affected retinal areas, resulting not only in absorption of soft drusen but also reduction or complete disappearance of drusenoid retinal pigment epithelium detachments. However, the character of the treatment process has raised suspicion that there is a decrease not only in cholesterol but also in antioxidants, such as vitamin E and vitamin A. METHODS: Twenty-three patients with the progressive dry form of age-related macular degeneration were each treated with 8 procedures of rheohaemapheresis. We measured levels of vitamin E (α-tocopherol), the vitamin E/cholesterol ratio in serum and lipoproteins (VLDL, LDL, HDL). Vitamin E in erythrocyte membrane and serum vitamin A (retinol) were also measured. These parameters were determined before and after rheohaemapheresis. Erythrocyte superoxide dismutase, erythrocyte glutathione peroxidase and serum malondialdehyde were analysed as markers of antioxidant activity and lipid peroxidation, respectively. RESULTS: In serum, the VLDL and LDL fraction ratios of vitamin E/cholesterol increased significantly. Additionally, the HDL fraction ratio showed an increase but this was not statistically significant. The patients showed no clinical signs of vitamin E deficiency, and their serum concentrations of vitamin E did not differ from normal values. The results show that rheohaemapheresis in addition to causing a significant reduction in atherogenic LDL cholesterol, may have favourable additive anti-atherogenic effects due to a relative increase in the content of vitamin E in the lipoprotein fractions.