Elevated Plasma Concentration of 4-Pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) Highlights Malignancy of Renal Cell Carcinoma.

Nicotinamide (NA) derivatives play crucial roles in various biological processes, such as inflammation, regulation of the cell cycle, and DNA repair. Recently, we proposed that 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR), an unusual derivative of NA, could be classified as an oncometabolite in bladder, breast, and lung cancer. In this study, we investigated the relations between NA metabolism and the progression, recurrence, metastasis, and survival of patients diagnosed with different histological subtypes of renal cell carcinoma (RCC). We identified alterations in plasma NA metabolism, particularly in the clear cell RCC (ccRCC) subtype, compared to papillary RCC, chromophobe RCC, and oncocytoma. Patients with ccRCC also exhibited larger tumor sizes and elevated levels of diagnostic serum biomarkers, such as hsCRP concentration and ALP activity, which were positively correlated with the plasma 4PYR. Notably, 4PYR levels were elevated in advanced stages of ccRCC cancer and were associated with a highly aggressive phenotype of ccRCC. Additionally, elevated concentrations of 4PYR were related to a higher likelihood of mortality, recurrence, and particularly metastasis in ccRCC. These findings are consistent with other studies, suggesting that NA metabolism is accelerated in RCC, leading to abnormal concentrations of 4PYR. This supports the concept of 4PYR as an oncometabolite and a potential prognostic factor in the ccRCC subtype.

Serum fatty acid profiles in breast cancer patients following treatment.

BACKGROUND: Breast cancer is associated with alterations in lipid metabolism. The treatment of breast cancer can also affect serum lipid composition. The purpose of this study was the examination of serum fatty acids (FAs) profiles in breast cancer survivors to assess if the FA levels normalize. METHODS: Serum levels of FAs were determined by gas chromatography-mass spectrometry in a group of breast cancer patients at baseline (before treatment, n = 28), at two follow-up visits at 12 months (n = 27) and 24 months (n = 19) after the breast cancer resection, and in the group of healthy controls (n = 25). Multivariate analysis was performed to assess how FA serum profile changes following treatment. RESULTS: Breast cancer patients' serum FA profiles at follow-ups did not normalize to the levels of control group. The greatest differences were found for levels of branched-chain (BCFA), odd-chain (OCFA) and polyunsaturated (PUFAs) FAs, all of which were significantly increased 12 months after the surgery. CONCLUSIONS: After treatment for breast cancer, the patients' serum FA profile differs from the profile before treatment and from controls, especially 12 months after treatment. Some changes may be beneficial - increased BCFA and OCFA levels, and improved n-6/n-3 PUFA ratio. This may reflect lifestyle changes in breast cancer survivors and have an impact on the risk of recurrence.

Rosiglitazone Ameliorates Cardiac and Skeletal Muscle Dysfunction by Correction of Energetics in Huntington's Disease.

Huntington's disease (HD) is a rare neurodegenerative disease that is accompanied by skeletal muscle atrophy and cardiomyopathy. Tissues affected by HD (central nervous system [CNS], skeletal muscle, and heart) are known to suffer from deteriorated cellular energy metabolism that manifests already at presymptomatic stages. This work aimed to test the effects of peroxisome proliferator-activated receptor (PPAR)-γ agonist-rosiglitazone on grip strength and heart function in an experimental HD model-on R6/1 mice and to address the mechanisms. We noted that rosiglitazone treatment lead to improvement of R6/1 mice grip strength and cardiac mechanical function. It was accompanied by an enhancement of the total adenine nucleotides pool, increased glucose oxidation, changes in mitochondrial number (indicated as increased citric synthase activity), and reduction in mitochondrial complex I activity. These metabolic changes were supported by increased total antioxidant status in HD mice injected with rosiglitazone. Correction of energy deficits with rosiglitazone was further indicated by decreased accumulation of nucleotide catabolites in HD mice serum. Thus, rosiglitazone treatment may not only delay neurodegeneration but also may ameliorate cardio- and myopathy linked to HD by improvement of cellular energetics.

Differences in Extracellular NAD+ and NMN Metabolism on the Surface of Vascular Endothelial Cells.

The disruption of the metabolism of extracellular NAD+ and NMN may affect related signaling cascades and pathologies, such as cardiovascular or respiratory system diseases. We aimed to study NAD+ and NMN hydrolysis on surface endothelial cells of diverse origins and with genetically modified nucleotide catabolism pathways. We tested lung endothelial cells isolated from C57BL/6 J wild-type (WT) and C57BL/6 J CD73 knockout (CD73 KO) mice, the transfected porcine iliac artery endothelial cell line (PIEC) with the human E5NT gene for CD73 (PIEC CD73), and a mock-transfected control (PIEC MOCK), as well as HMEC-1 and H5V cells. Substrate conversion into the product was followed by high-performance liquid chromatography (HPLC). We showed profound differences in extracellular NAD+ and NMN metabolism related to the vessel origin, species diversity, and type of culture. We also confirmed the involvement of CD38 and CD73 in NAD+ and NMN cleavage.

The comparison of nucleotide metabolites and amino acids patterns in patients with eating disorders, with and without symptoms of depression.

Purines, pyrimidines, and amino acid level have gained attention recently as potential determinants of mental disorders. However, eating disorders patients (ED) have not been yet appropriately studied, especially subjects with coexisting mood disorders. This paper examines the serum level of nucleotide catabolites and plasma amino acids in eating disorders with hyperphagia, with and without Major Depressive Disorder (MDD). Samples were taken from adult persons suffering from eating disorders (two forms: simple obesity and binge eating disorder) with MDD (n = 20) and without (n = 17). Serum nucleotides and plasma amino acids concentrations were analyzed with high-performance liquid chromatography-mass spectrometry. The nucleotides metabolite in MDD patients had a significantly (p < 0.05) lower uridine. Among MDD patients with ED significantly (p < 0.05) higher levels of asparagine, glutamine, proline, and arginine were found as compared to the control group. This study demonstrated differences in nucleotide metabolite and amino acid pattern in depression patients with eating disorders. This may be relevant to the mechanisms and may help identify biomarkers.

The decreased serum activity of cytosolic 5'-nucleotidase IA as a potential marker of breast cancer-associated muscle inflammation.

Cytosolic 5'-nucleotidase IA (cN-IA) plays a central role in the regulation of the purine nucleotide pool in skeletal muscle, preferentially converting adenosine monophosphate to adenosine. cN-IA can act as an autoantigen in muscle diseases, including the paraneoplastic syndrome related to breast cancer (BC). As a result of myocyte damage, released cN-IA protein may trigger the production of anti-cN-IA antibodies (anti-NT5C1A). This work aimed to develop an effective method to measure cN-IA activity in the serum and analyze it in BC patients. Our study demonstrated that serum cN-IA activity was decreased in BC patients and we assumed it is due to the presence of specific autoantibodies. We found correlations between cN-IA activity and parameters of inflammatory muscle damage. Thus, cN-IA is worth further attention to clarify its usefulness as a biomarker of BC-associated polymyositis.

Enhanced Muscle Strength in Dyslipidemic Mice and Its Relation to Increased Capacity for Fatty Acid Oxidation.

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR -/-) leads to improvement of exercise capacity. This study aimed to investigate in detail skeletal muscle function and metabolism in these dyslipidemic mice. We found that ApoE/LDLR -/- mice showed an increased grip strength as well as increased troponins, and Mhc2 levels in skeletal muscle. It was accompanied by the increased skeletal muscle mitochondria numbers (judged by increased citrate synthase activity) and elevated total adenine nucleotides pool. We noted increased triglycerides contents in skeletal muscles and increased serum free fatty acids (FFA) levels in ApoE/LDLR -/- mice. Importantly, Ranolazine mediated inhibition of FFA oxidation in ApoE/LDLR -/- mice led to the reduction of exercise capacity and total adenine nucleotides pool. Thus, this study demonstrated that increased capacity for fatty acid oxidation, an adaptive response to dyslipidemia leads to improved cellular energetics that translates to increased skeletal muscle strength and contributes to increased exercise capacity in ApoE/LDLR -/- mice.

An unusual nicotinamide derivative, 4-pyridone-3-carboxamide ribonucleoside (4PYR), is a novel endothelial toxin and oncometabolite.

Our recent studies identified a novel pathway of nicotinamide metabolism that involves 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) and demonstrated its endothelial cytotoxic effect. This study tested the effects of 4PYR and its metabolites in experimental models of breast cancer. Mice were divided into groups: 4T1 (injected with mammary 4T1 cancer cells), 4T1 + 4PYR (4PYR-treated 4T1 mice), and control, maintained for 2 or 21 days. Lung metastasis and endothelial function were analyzed together with blood nucleotides (including 4PYR), plasma amino acids, nicotinamide metabolites, and vascular ectoenzymes of nucleotide catabolism. 4PYR metabolism was also evaluated in cultured 4T1, MDA-MB-231, MCF-7, and T47D cells. An increase in blood 4PYR in 4T1 mice was observed at 2 days. 4PYR and its metabolites were noticed after 21 days in 4T1 only. Higher blood 4PYR was linked with more lung metastases in 4T1 + 4PYR vs. 4T1. Decreased L-arginine, higher asymmetric dimethyl-L-arginine, and higher vascular ecto-adenosine deaminase were observed in 4T1 + 4PYR vs. 4T1 and control. Vascular relaxation caused by flow-dependent endothelial activation in 4PYR-treated mice was significantly lower than in control. The permeability of 4PYR-treated endothelial cells was increased. Decreased nicotinamide but enhanced nicotinamide metabolites were noticed in 4T1 vs. control. Reduced N-methylnicotinamide and a further increase in Met2PY were observed in 4T1 + 4PYR vs. 4T1 and control. In cultured breast cancer cells, estrogen and progesterone receptor antagonists inhibited the production of 4PYR metabolites. 4PYR formation is accelerated in cancer and induces metabolic disturbances that may affect cancer progression and, especially, metastasis, probably through impaired endothelial homeostasis. 4PYR may be considered a new oncometabolite.

The new insight into extracellular NAD+ degradation-the contribution of CD38 and CD73 in calcific aortic valve disease.

Nicotinamide adenine dinucleotide (NAD+ ) is crucial for cell energy metabolism and many signalling processes. Recently, we proved the role of ecto-enzymes in controlling adenine nucleotide-dependent pathways during calcific aortic valve disease (CAVD). This study aimed to investigate extracellular hydrolysis of NAD+ and mononucleotide nicotinamide (NMN) in aortic valves and aorta fragments of CAVD patients and on the inner aortic surface of ecto-5'-nucleotidase knockout mice (CD73-/-). Human non-stenotic valves (n = 10) actively converted NAD+ and NMN via both CD73 and NAD+ -glycohydrolase (CD38) according to our analysis with RP-HPLC and immunofluorescence. In stenotic valves (n = 50), due to reduced CD73 activity, NAD+ was degraded predominantly by CD38 and additionally by ALP and eNPP1. CAVD patients had significantly higher hydrolytic rates of NAD+ (0.81 ± 0.07 vs 0.56 ± 0.10) and NMN (1.12 ± 0.10 vs 0.71 ± 0.08 nmol/min/cm2 ) compared with controls. CD38 was also primarily engaged in human vascular NAD+ metabolism. Studies using specific ecto-enzyme inhibitors and CD73-/- mice confirmed that CD73 is not the only enzyme involved in NAD+ and NMN hydrolysis and that CD38 had a significant contribution to these pathways. Modifications of extracellular NAD+ and NMN metabolism in aortic valve cells may be particularly important in valve pathology and could be a potential therapeutic target.

Macrophage-Derived Adenosine Deaminase 2 Correlates with M2 Macrophage Phenotype in Triple Negative Breast Cancer.

Several lines of evidence suggest that altered adenosine deaminase (ADA) activity, especially its ADA2 iso-enzyme, is associated with malignant breast cancer (BC) development. Triple-negative breast cancer (TNBC) is currently the most challenging BC subtype due to its metastatic potential and recurrence. Herein, we analyzed the sources of ADA iso-enzymes in TNBC by investigating the effects of cell-to-cell interactions between TNBC cells, macrophages, lymphocytes, and endothelial cells. We also examined the potential relationship between ADA activity and cancer progression in TNBC patients. In vitro analyses demonstrated that the interactions of immune and endothelial cells with MDA-MB-231 triple negative BC cells modulated their extracellular adenosine metabolism pattern. However, they caused an increase in the ADA1 activity, and did not alter ADA2 activity in cancer cells. In turn, the co-culture of MDA-MB-231 cells with THP-1 monocyte/macrophages, Jurkat cells, and human lung microvascular endothelial cells (HULEC) caused the increase in ADA2 activity on THP-1 cells and ADA1 activity on Jurkat cells and HULEC. Clinical sample analysis revealed that TNBC patients had higher plasma ADA2 activities and lower ADA1/ADA2 ratio at advanced stages of cancer development than in the initial stages, while patients with hormone receptor positive, HER2 negative (HR+HER2-), and triple positive (HR+HER2+) breast cancers at the same stages showed opposite trends. TNBC patients also demonstrated positive associations between plasma ADA2 activity and pro-tumor M2 macrophage markers, as well as between ADA1 activity and endothelial dysfunction or inflammatory parameters. The analysis of TNBC patients, at 6 and 12 months following cancer treatment, did not showed significant changes in plasma ADA activities and macrophage polarization markers, which may be the cause of their therapeutic failure. We conclude that alterations in both ADA iso-enzymes can play a role in breast cancer development and progression by the modulation of extracellular adenosine-dependent pathways. Additionally, the changes in ADA2 activity that may contribute to the differentiation of macrophages into unfavorable pro-tumor M2 phenotype deserve special attention in TNBC.

Comparison of plasma nucleotide metabolites and amino acids pattern in patients with binge eating disorder and obesity.

Binge eating disorder (BED) increasingly affects population, but the mechanisms of the disease and its biomarkers are not well characterized. Recently, plasma purines, pyrimidines, amino acid and nicotinamide metabolites profiling attracted attention in studies on pathology and biomarkers of mental disorders but has not been adequately studied in BED. Blood and plasma samples were taken from patients with adult obese with BED (n = 20) and control adult obese without BED (n = 17). Plasma samples were analyzed for nucleotides and amino acid concentrations with high-performance liquid chromatography-mass spectrometry. BED had a significantly (p < 0.05) lower uridine and hypoxanthine to creatinine ratio compared to the control group. Among the amino acids BED patients had significantly (p < 0.05) lower concentrations of glutamic acid, leucine, isoleucine and the whole branched-chain amino acids group, while the concentration of citrulline was increased. Among nicotinamide metabolites, 1-methylnicotinamide levels were significantly (p < 0.05) lower. This study highlights potential use of profiling nucleotide metabolite and amino acid pattern in BED patients that may provide information on mechanisms and potential biomarkers. However, further investigation in larger population is necessary to identify clinical correlates of the observed changes.

Statin treatment of patients with calcific aortic valve disease modulates extracellular adenosine metabolism on the cell surface of the aortic valve.

Statins efficiently prevent cardiovascular events by lipid-dependent and independent mechanisms. We hypothesize that part of these protective effects could be associated with an increased extracellular adenosine signaling. We demonstrated previously that aortic valves obtained from patients with calcific aortic valve disease (CAVD) disclosed disturbances in extracellular adenosine metabolism. This study aimed to analyze the impact of statin treatment on extracellular nucleotides and adenosine metabolism in aortic valves originated from CAVD patients and to elucidate potential mechanisms that are involved in the regulation of ecto-enzyme activities by statins. Aortic valves of CAVD patients treated with statins (n = 45) revealed higher adenosine production and its lower degradation than in non-treated patients (n = 28). Statin treatment was also related to the improvement in pre-operative echocardiographic data indicating milder aortic valve stenosis and a better function of the left ventricle. The rates of aortic valve adenosine conversions correlated with plasma lipid profile parameters, within both statin-treated and non-treated groups. Valvular extracellular AMP hydrolysis correlated negatively, while adenosine deamination positively with plasma total and LDL cholesterol. Atorvastatin treatment of murine heart endothelial cells led to the enhanced ecto-5'nucleotidase (CD73) and decreased ecto-adenosine deaminase (eADA) activity. When endothelial cells were stimulated with thrombin that induces endothelial cell exocytosis, activities of both cell-surface CD73 and eADA were increased, while co-treatment with atorvastatin reversed only thrombin-induced eADA activity. In conclusion, early intervention with statins may provide beneficial effects for CAVD therapy. Here, we presented results showing that these protective outcomes could be mediated via the regulation of extracellular adenosine metabolism pathways.

Systemic Effects of Radiotherapy and Concurrent Chemo-Radiotherapy in Head and Neck Cancer Patients-Comparison of Serum Metabolome Profiles.

Anticancer treatment induces systemic molecular changes that could be detected at the level of biofluids. Understanding how human metabolism is influenced by these treatments is crucial to predict the individual response and adjust personalized therapies. Here, we aimed to compare profiles of metabolites in serum of head and neck cancer patients treated with concurrent chemo-radiotherapy, radiotherapy alone, or induction chemotherapy. Serum samples were analyzed by a targeted quantitative approach using combined direct flow injection and liquid chromatography coupled to tandem mass spectrometry, which allowed simultaneous quantification of 149 metabolites. There were 45 metabolites whose levels were significantly changed between pretreatment and within- or post-treatment serum samples, including 38 phospholipids. Concurrent chemo-radiotherapy induced faster and stronger effects than radiotherapy alone. On the other hand, chemotherapy alone did not result in significant changes. The decreased level of total phospholipids was the most apparent effect observed during the first step of the treatment. This corresponded to the loss of patients' body mass, yet no correlation between both parameters was observed for individual patients. We concluded that different molecular changes were measured at the level of serum metabolome in response to different treatment modalities.

Nucleotide ecto-enzyme metabolic pattern and spatial distribution in calcific aortic valve disease; its relation to pathological changes and clinical presentation.

BACKGROUND: Extracellular nucleotide metabolism contributes to chronic inflammation, cell differentiation, and tissue mineralization by controlling nucleotide and adenosine concentrations and hence its purinergic effects. This study investigated location-specific changes of extracellular nucleotide metabolism in aortic valves of patients with calcific aortic valve disease (CAVD). Individual ecto-enzymes and adenosine receptors involved were analyzed together with correlation with CAVD severity and risk factors. RESULTS: Nucleotide and adenosine degradation rates were adversely modified on the aortic surface of stenotic valve as compared to ventricular side, including decreased ATP removal (1.25 ± 0.35 vs. 2.24 ± 0.61 nmol/min/cm2) and adenosine production (1.32 ± 0.12 vs. 2.49 ± 0.28 nmol/min/cm2) as well as increased adenosine deamination (1.28 ± 0.31 vs. 0.67 ± 0.11 nmol/min/cm2). The rates of nucleotide to adenosine conversions were lower, while adenosine deamination was higher on the aortic sides of stenotic vs. non-stenotic valve. There were no differences in extracellular nucleotide metabolism between aortic and ventricular sides of non-stenotic valves. Furthermore, nucleotide degradation rates, measured on aortic side in CAVD (n = 62), negatively correlated with echocardiographic and biochemical parameters of disease severity (aortic jet velocity vs. ATP hydrolysis: r = - 0.30, p < 0.05; vs. AMP hydrolysis: r = - 0.44, p < 0.001; valvular phosphate concentration vs. ATP hydrolysis: r = - 0.26, p < 0.05; vs. AMP hydrolysis: r = - 0.25, p = 0.05) while adenosine deamination showed positive correlation trend with valvular phosphate deposits (r = 0.23, p = 0.07). Nucleotide and adenosine conversion rates also correlated with CAVD risk factors, including hyperlipidemia (AMP hydrolysis vs. serum LDL cholesterol: r = - 0.28, p = 0.05; adenosine deamination vs. total cholesterol: r = 0.25, p = 0.05; LDL cholesterol: r = 0.28, p < 0.05; triglycerides: r = 0.32, p < 0.05), hypertension (adenosine deamination vs. systolic blood pressure: r = 0.28, p < 0.05) and thrombosis (ATP hydrolysis vs. prothrombin time: r = - 0.35, p < 0.01). Functional assays as well as histological and immunofluorescence, flow cytometry and RT-PCR studies identified all major ecto-enzymes engaged in nucleotide metabolism in aortic valves that included ecto-nucleotidases, alkaline phosphatase, and ecto-adenosine deaminase. We have shown that changes in nucleotide-converting ecto-enzymes were derived from their altered activities on valve cells and immune cell infiltrate. We have also demonstrated a presence of A1, A2a and A2b adenosine receptors with diminished expression of A2a and A2b in stenotic vs. non-stenotic valves. Finally, we revealed that augmenting adenosine effects by blocking adenosine deamination with deoxycoformycin decreased aortic valve thickness and reduced markers of calcification via adenosine-dependent pathways in a mouse model of CAVD. CONCLUSIONS: This work highlights profound changes in extracellular nucleotide and adenosine metabolism in CAVD. Altered extracellular nucleotide hydrolysis and degradation of adenosine in stenotic valves may affect purinergic responses to support a pro-stenotic milieu and valve calcification. This emphasizes a potential mechanism and target for prevention and therapy. .

The Effect of One Anastomosis Gastric Bypass on Branched-Chain Fatty Acid and Branched-Chain Amino Acid Metabolism in Subjects with Morbid Obesity.

BACKGROUND: Subjects with morbid obesity have low levels of serum branched-chain fatty acids (BCFAs), which correlate inversely with insulin resistance, hypertriglyceridemia, and inflammation. Recent evidence suggests BCFAs are produced during branched-chain amino acid (BCAA) catabolism in human adipose tissue. Elevated concentrations of BCAAs are associated with insulin resistance. OBJECTIVES: In this single-center study, we evaluated the effect of one anastomosis gastric bypass (OAGB) on circulating BCFA and BCAA. Moreover, we determined the expression of genes involved in BCAA catabolism in adipose tissue of patients with obesity and lean controls. METHODS: Fasting levels of BCFAs and BCAAs were determined by gas and liquid chromatography, respectively, coupled with mass spectrometry, in 50 patients with morbid obesity before and 6-9 months after surgery, and in 32 lean controls. Visceral and subcutaneous adipose tissue (VAT and SAT, respectively) biopsies were collected at baseline to determine mRNA levels for enzymes involved in BCAA catabolism. RESULTS: Before surgery, patients with obesity had lower BCFAs and greater BCAAs than control subjects. OAGB increased BCFA and decreased BCAA levels. Insulin resistance (assessed by HOMA) correlated inversely with BCFAs and positively with BCAAs. Expression of genes involved in BCAA catabolism in VAT (but not SAT) was lower in patients with obesity than in lean controls. CONCLUSIONS: OAGB-induced weight loss increases circulating BCFAs and decreases circulating BCAAs in patients with morbid obesity, perhaps by altering BCAA catabolism in VAT. We speculate that this shift may be related to the improvement in insulin sensitivity after surgery.

Increased plasma concentration of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) in lung cancer. Preliminary studies.

4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) is a new nicotinamide derivative, which is potentially toxic to the endothelium. Dysfunction of the endothelium promotes cancer cell proliferation, invasiveness, and inflammatory signaling. The aim of this study was to analyze 4PYR concentration in the plasma of lung cancer patients and its relationship to other known biochemical parameters associated with the endothelium function. The concentration of 4PYR, nicotinamide, 1-methylnicotinamide (MNA), amino acids, and their derivatives were measured in samples obtained from patients with primary squamous cell carcinoma (n = 48) and control group (n = 100). The concentration of 4PYR and 4PYR/MNA ratio were significantly higher in lung cancer patients as compared to controls (0.099 ± 0.009 vs. 0.066 ± 0.006 µmol/L and 1.10 ± 0.08 vs. 1.97 ± 0.15, respectively). The plasma arginine/asymmetric dimethylarginine (Arg/ADMA) ratio was considerably lower in lung cancer patients (253 ± 17 vs. 369 ± 19) as well as plasma MNA (0.057 ± 0.004 vs. 0.069 ± 0.003 µmol/L). There was no difference in the plasma concentrations of nicotinamide and nicotinamide riboside in both groups (0.116 ± 0.019 vs. 0.131 ± 0.014 and 0.102 ± 0.006 vs. 0.113 ± 0.011, respectively). In this study, a higher 4PYR concentration was observed for the first time in patients with squamous cell carcinoma. This change may be related to the endothelial dysfunction that promote cancer progression since 4PYR and its derivatives are known to disrupt glycolytic pathway.

Inhibition of LPS-stimulated ecto-adenosine deaminase attenuates endothelial cell activation.

Vascular inflammation is an important factor in the pathophysiology of cardiovascular diseases, such as atherosclerosis. Changes in the extracellular nucleotide and in particular adenosine catabolism may alter a chronic inflammation and endothelial activation. This study aimed to evaluate the relation between vascular ecto-adenosine deaminase (eADA) activity and endothelial activation in humans and to analyze the effects of LPS-mediated inflammation on this activity as well as mechanisms of its increase. Moreover, we investigated a therapeutic potential of ADA inhibition by deoxycofromycin (dCF) for endothelial activation. We demonstrated a positive correlation of vascular eADA activity and ADA1 mRNA expression with endothelial activation parameters in humans with atherosclerosis. The activation of vascular eADA was also observed under LPS stimulation in vivo along with endothelial activation, an increase in markers of inflammation and alterations in the lipid profile of a rat model. Ex vivo and in vitro studies on human specimen demonstrated that at an early stage of vascular pathology, eADA activity originated from activated endothelial cells, while at later stages also from an inflammatory infiltrate. We proposed that LPS-stimulated increase in endothelial adenosine deaminase activity could be a result of IL-6/JAK/STAT pathway activation, since the lack of IL-6 in mice was associated with lower vascular and plasma eADA activities. Furthermore, the inhibitors of JAK/STAT pathway decreased LPS-stimulated adenosine deaminase activity in endothelial cells. We demonstrated that cell surface eADA activity could be additionally regulated by transcytosis pathways, as exocytosis inhibitors including lipid raft inhibitor, methyl-ß-cyclodextrin decreased LPS-induced eADA activity. This suggests that cholesterol-dependent protein externalization mediated by lipid rafts could be an important factor in the eADA increase. Moreover, endocytosis inhibitors and exocytosis activators increased this activity on the cell surface. Furthermore, the inhibition of adenosine deaminase in endothelial cells in vitro attenuated LPS-mediated IL-6 release and soluble ICAM-1 and VCAM-1 concentration in the incubation medium through the restoration of the extracellular adenosine pool and adenosine receptor-dependent pathways. This study demonstrated that the vascular endothelial eADA activity remains under control of inflammatory mediators acting through JAK/STAT pathway that could be further modified by dyslipidemic-dependent exocytosis and transcytosis pathways. Inhibition of eADA blocked endothelial activation suggesting a crucial role of this enzyme in the control of vascular inflammation. This supports the concept of eADA targeted vascular protection therapy.

One Anastomosis Gastric Bypass Reconstitutes the Appropriate Profile of Serum Amino Acids in Patients with Morbid Obesity.

Bariatric surgery leads to metabolic benefits in patients with obesity, but their mechanisms are not well understood. The appropriate composition of serum amino acids (AA) is important for sufficient supply of these components into various tissues and organs. Obesity leads to alterations in serum AA concentrations. The aim of this study was to examine the effect of one anastomosis gastric bypass (OAGB), a promising type of bariatric surgery, on serum AA concentrations, which were assayed by LC-MS in serum of 46 bariatric patients prior to and 6-9 months after OAGB, as well as in 30 lean control subjects. The results were analyzed by principle components analysis and metabolic pathway analysis. PCA analysis showed that OAGB led to normalization of serum AA concentrations of patients with obesity to a pattern similar to the control subjects, and the concentrations of essential AA remained decreased after OAGB. Changes of individual AA and their associated metabolic pathways were also presented. OAGB caused normalization of the AA profile, which may contribute to improvement of glucose homeostasis and reduction of cardiovascular risk. Considering decreased essential AA concentrations after OAGB, increased intake of high protein food should be recommended to the patients after this type of bariatric surgery.

Simultaneous accurate quantification of HO-1, CD39, and CD73 in human calcified aortic valves using multiple enzyme digestion - filter aided sample pretreatment (MED-FASP) method and targeted proteomics.

Several proteins such as membrane-associated ectonucleotidases: ecto-5'-nucleotidase (E5NT/CD73) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), and intracellular heme oxygenase-1 (HO-1) may contribute to protection from inflammation-related diseases such as calcific aortic valve stenosis (CAS). Accurate quantification of these proteins could contribute to better understanding of the disease mechanisms and identification of biomarkers. This report presents development and validation of quantification method for E5NT/CD73, ENTPD1/CD39 and HO-1. The multiplexed targeted proteomic assay involved antibody-free, multiple-enzyme digestion, filter-assisted sample preparation (MED-FASP) strategy and a nanoflow liquid chromatography/mass spectrometry under multiple reaction monitoring mode (LC-MRM/MS). The method developed presented high sensitivity (LLOQ of 5 pg/mL for each of the analytes) and accuracy that ranged from 92.0% to 107.0%, and was successfully applied for the absolute quantification of HO-1, CD39 and CD73 proteins in homogenates of human calcified and non-calcified valves. The absolute CD39 and CD73 concentrations were lower in calcified aortic valves (as compared to non-stenotic ones) and were found to be: 1.16 ± 0.39 vs. 3.15 ± 0.37 pmol/mg protein and 1.94 ± 0.21 vs. 2.39 ± 0.39 pmol/mg protein, respectively, while the quantity of HO-1 was elevated in calcified valves (10.72 ± 1.18 vs. 4.28 ± 0.42 amol/mg protein). These results were consistent but more reproducible as compared to immunoassays. In conclusion, multiplexed quantification of HO-1, CD39 and CD73 proteins by LC-MRM/MS works well in challenging human tissues such as aortic valves. This analysis confirmed the relevance of these proteins in pathogenesis of CAS and could be extended to other biomedical investigations.

Nicotinamide metabolism alterations in bladder cancer: Preliminary studies.

OBJECTIVES: Cancer is one of the main cause of death in Western countries. Inflammation plays an important role in the pathogenesis of cancer. Nicotinamide (NA) - known for its anti-inflammatory properties - participates in the processes related to the cell cycle regulation and DNA repair, which are relevant in cancer development. This study aimed to investigate the nicotinamide metabolism alterations in bladder cancer. METHODS: Blood and plasma samples of patients with bladder cancer were collected. Blood pyridine and adenine nucleotides concentration were measured using high performance liquid chromatography (HPLC). Plasma nicotinamide metabolites concentration were determined using high performance liquid chromatography - mass spectrometry (LC/MS). RESULTS: Our results indicated that the development of bladder cancer caused significant decrease in the concentration of N-methylnicotinamide (MetNA) (0.07 ± 0.02 vs 0.1 ± 0.03 µmol/l) and an increase in the concentration of N-methyl-2-pyridone-5-carboxamide (Met2PY) - one of the final nicotinamide metabolites: (1.1 ± 0.15 vs 0.7 ± 0.07 µmol/l) in comparison to the control. The association between the stage of cancer and the increase in both, Met2PY and Met4PY levels was observed. Blood ATP and NAD levels were significantly decreased in bladder cancer patients as compared to the control (970.8 ± 77.84 vs 1165.00 ± 57.76 µmol/l; 45.86 ± 2.98 vs 53.06 ± 2.28 µmol/l respectively). CONCLUSIONS: Bladder cancer development caused substantial changes in nicotinamide metabolism, such as decreased plasma MetNA and increased Met2PY concentration. Analysis of the nicotinamide and its metabolites concentrations - as new biomarkers - may allow to track the course of pathological processes in cancer.