Novel mass spectrometry-based assay for thymidylate synthase activity.

Thymidylate synthase (TS) is an enzyme responsible for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), with the co-substrate 5,10-methylenetetrahydrofolate (5,10-CH2-THF) as the methyl donor. TS is the only enzyme capable of de novo biosynthesis of dTMP in humans, a nucleotide crucial for DNA synthesis and therefore cell proliferation and survival. As such, TS is a major drug target in chemotherapy by compounds such as 5-fluorouracil. Due to the clinical and physiological importance of TS, the ability to accurately assay its activity is crucial. Several assays have been developed for this purpose, relying on spectrophotometry or radioisotope labeling methods. In this study, we have developed a liquid chromatography - mass spectrometry-based method for assessing TS activity by direct and specific measurement of the reaction product, dTMP. The assay was tested on mouse liver homogenates. We noted that excessive 5,10-CH2-THF concentration (400 µM) led to substrate inhibition and therefore 200 µM was used. The activity assayed at 1 µM dUMP was linear with protein content and time (up to 60 min) and was 0.56 ± 0.12 pmol/mg protein/min, in line with previously reported values. Additionally, by using a high mass resolution Orbitrap instrument side reactions were monitored, revealing major changes in folate pools and nucleotide metabolism. These findings highlight the value of the developed TS assay for routine TS activity monitoring in complex matrixes such as clinical material.

The Physiological and Pathological Role of Acyl-CoA Oxidation.

Fatty acid metabolism, including ß-oxidation (ßOX), plays an important role in human physiology and pathology. ßOX is an essential process in the energy metabolism of most human cells. Moreover, ßOX is also the source of acetyl-CoA, the substrate for (a) ketone bodies synthesis, (b) cholesterol synthesis, (c) phase II detoxication, (d) protein acetylation, and (d) the synthesis of many other compounds, including N-acetylglutamate-an important regulator of urea synthesis. This review describes the current knowledge on the importance of the mitochondrial and peroxisomal ßOX in various organs, including the liver, heart, kidney, lung, gastrointestinal tract, peripheral white blood cells, and other cells. In addition, the diseases associated with a disturbance of fatty acid oxidation (FAO) in the liver, heart, kidney, lung, alimentary tract, and other organs or cells are presented. Special attention was paid to abnormalities of FAO in cancer cells and the diseases caused by mutations in gene-encoding enzymes involved in FAO. Finally, issues related to α- and ω- fatty acid oxidation are discussed.

The Role of Acyl-CoA ß-Oxidation in Brain Metabolism and Neurodegenerative Diseases.

This review highlights the complex role of fatty acid ß-oxidation in brain metabolism. It demonstrates the fundamental importance of fatty acid degradation as a fuel in energy balance and as an essential component in lipid homeostasis, brain aging, and neurodegenerative disorders.

Alterations in the amino acid profile in patients with papillary thyroid carcinoma with and without Hashimoto's thyroiditis.

Purpose: Amino acids (AAs) play important physiological roles in living cells. Some amino acid changes in blood are specific for autoimmune disorders, and some are specific for thyroid cancer. The aims of this study were to profile AA metabolites in the serum of patients with papillary thyroid carcinoma (PTC0) without Hashimoto's thyroiditis (HT) and patients with PTC with HT (PTC1) and predict whether AA metabolites are associated with thyroid disease, thyroid hormone and thyroid autoantibodies. Methods: A total of 95 serum samples were collected, including 28 healthy controls (HCs), 28 PTC0 patients and 39 PTC1 patients. Serum samples were analyzed by high-performance liquid chromatography-triple stage quadrupole-mass spectrometry (HPLC-TSQ-MS), and twenty-one amino acids (AAs) were detected. Results: The serum concentration of glutamic acid was significantly elevated in PTC1 patients compared with PTC0 patients. Lysine was the second amino acid that differentiated these two groups of PTC patients. In addition, the serum concentrations of glycine, alanine and tyrosine were significantly reduced in both PTC patient groups compared to the HC group. These AAs were also correlated with thyroid hormones and antibodies. Five amino acid markers, namely, glycine, tyrosine, glutamic acid, glutamine and arginine, separated/distinguished PTC0 patients from healthy subjects, and eight AA markers, the same AAs as above without arginine but with alanine, leucine, valine and histidine, separated/distinguished PTC1 patients from healthy subjects based on ROC analysis. Conclusion: Compared with the HCs, changes in AAs in PTC0 and PTC1 patients showed similar patterns, suggesting the possibility of a common pathophysiological basis, which confirms preliminary research that PTC is significantly associated with pathologically confirmed HT. We found two AAs, lysine and alanine, that can perform diagnostic functions in distinguishing PTC1 from PTC0.

Effect of Hyperbaric Oxygenation on Blood Cytokines and Arginine Derivatives; No Evidence for Induction of Inflammation or Endothelial Injury.

(1) Background: Hyperbaric oxygen therapy (HBOT) uses 100% oxygen delivered at 1.5-3 times the atmospheric pressure in a specialised chamber to achieve supraphysiological oxygen tension in blood and tissues. Besides its target, HBOT may affect inflammation, endothelial function or angiogenesis. This study analysed the effect of HBOT on blood concentrations of factors that may affect these processes in patients with necrotizing soft-tissue infections (NSTI), aseptic bone necrosis (ABN) and idiopathic sudden sensory neural hearing loss (ISSNHL). (2) Methods: Concentrations asymmetric dimethylarginine (ADMA) and other arginine derivatives were measured with liquid chromatography/mass spectrometry, whereas ELISA was used to quantitate vascular endothelial growth factor (VEGF) and cytokines (IL-1, IL-4, IL-6, IL-10, TGF-ß) before and after HBOT in 80 patients (NSTI n = 21, ISSNHL n = 53, ABN n = 6). (3) Results: While some differences were noted between patient groups in ADMA and other arginine derivatives as well as in cytokine concentrations, HBOT did not affect any of these parameters. (4) Conclusions: While cytokines and arginine derivatives concentrations were modified by underlying pathology, hyperbaric oxygenation did not immediately modify it suggesting that it is neutral for inflammation and is not inducing endothelial injury.

Lipid Alterations in Systemic Sclerosis.

Background: Systemic sclerosis (SSc) is an autoimmune disease with an elusive etiology and poor prognosis. Due to its diverse clinical presentation, a personalized approach is obligatory and needs to be based on a comprehensive biomarker panel. Therefore, particular metabolomic studies are necessary. Lipidomics addressed these issues and found disturbances in several crucial metabolic pathways. Aim of Review: The review aims to briefly summarize current knowledge related to lipid alterations in systemic sclerosis, highlight its importance, and encourage further research in this field. Key Scientific Concepts of Review: In this review, we summarized the studies on the lipidomic pattern, fatty acids, lipoproteins, cholesterol, eicosanoids, prostaglandins, leukotrienes, lysophospholipids, and sphingolipids in systemic sclerosis. Researchers demonstrated several alternate aspects of lipid metabolism. As we aimed to present our findings in a comprehensive view, we decided to divide our findings into three major groups: "serum lipoproteins," "fatty acids and derivatives," and "cellular membrane components," as we do believe they play a prominent role in SSc pathology.

The Pathophysiological Role of CoA.

The importance of coenzyme A (CoA) as a carrier of acyl residues in cell metabolism is well understood. Coenzyme A participates in more than 100 different catabolic and anabolic reactions, including those involved in the metabolism of lipids, carbohydrates, proteins, ethanol, bile acids, and xenobiotics. However, much less is known about the importance of the concentration of this cofactor in various cell compartments and the role of altered CoA concentration in various pathologies. Despite continuous research on these issues, the molecular mechanisms in the regulation of the intracellular level of CoA under pathological conditions are still not well understood. This review summarizes the current knowledge of (a) CoA subcellular concentrations; (b) the roles of CoA synthesis and degradation processes; and (c) protein modification by reversible CoA binding to proteins (CoAlation). Particular attention is paid to (a) the roles of changes in the level of CoA under pathological conditions, such as in neurodegenerative diseases, cancer, myopathies, and infectious diseases; and (b) the beneficial effect of CoA and pantethine (which like CoA is finally converted to Pan and cysteamine), used at pharmacological doses for the treatment of hyperlipidemia.

Metallothioneins 1 and 2, but not 3, are regulated by nutritional status in rat white adipose tissue.

BACKGROUND: Cumulating evidence underlines the role of adipose tissue metallothionein (MT) in the development of obesity and type 2 diabetes. Fasting/refeeding was shown to affect MT gene expression in the rodent liver. The influence of nutritional status on MT gene expression in white adipose tissue (WAT) is inconclusive. The aim of this study was to verify if fasting and fasting/refeeding may influence expression of MT genes in WAT of rats. RESULTS: Fasting resulted in a significant increase in MT1 and MT2 gene expressions in retroperitoneal, epididymal, and inguinal WAT of rats, and this effect was reversed by refeeding. Altered expressions of MT1 and MT2 genes in all main fat depots were reflected by changes in serum MT1 and MT2 levels. MT1 and MT2 messenger RNA (mRNA) levels in WAT correlated inversely with serum insulin concentration. Changes in MT1 and MT2 mRNA levels were apparently not related to total zinc concentrations and MTF1 and Zn transporter mRNA levels in WAT. Fasting or fasting/refeeding exerted no effect on the expression of MT3 gene in WAT. Addition of insulin to isolated adipocytes resulted in a significant decrease in MT1 and MT2 gene expressions. In contrast, forskolin or dibutyryl-cAMP (dB-cAMP) enhanced the expressions of MT1 and MT2 genes in isolated adipocytes. Insulin partially reversed the effect of dB-cAMP on MT1 and MT2 gene expressions. CONCLUSIONS: This study showed that the expressions of MT1 and MT2 genes in WAT are regulated by nutritional status, and the regulation may be independent of total zinc concentration.

Effect of AMP-deaminase 3 knock-out in mice on enzyme activity in heart and other organs.

Recent findings suggest that inhibition of AMP-deaminase (AMPD) could be effective therapeutic strategy in heart disease associated with cardiac ischemia. To establish experimental model to study protective mechanisms of AMPD inhibition we developed conditional, cardiac specific knock-outs in Cre recombinase system. AMPD3 floxed mice were crossed with Mer-Cre-Mer mice. Tamoxifen was injected to induce Cre recombinase. After two weeks, hearts, skeletal muscle, liver, kidney, and blood were collected and activities of AMPD and related enzymes were analyzed using HPLC-based procedure. We demonstrate loss of more than 90% of cardiac AMPD activity in the heart of AMPD3-/-mice while other enzymes of nucleotide metabolism such as adenosine deaminase, purine nucleoside phosphorylase were not affected. Surprisingly, activity of AMPD was also reduced in the erythrocytes and in the kidney by 20%-30%. No change of AMPD activity was observed in the skeletal muscle and the liver.

AMP deaminase 1 gene polymorphism and heart disease-a genetic association that highlights new treatment.

Nucleotide metabolism and signalling is directly linked to myocardial function. Therefore analysis how diversity of genes coding nucleotide metabolism related proteins affects clinical progress of heart disease could provide valuable information for development of new treatments. Several studies identified that polymorphism of AMP deaminase 1 gene (AMPD1), in particular the common C34T variant of this gene was found to benefit patients with heart failure and ischemic heart disease. However, these findings were inconsistent in subsequent studies. This prompted our detailed analysis of heart transplant recipients that revealed diverse effect: improved early postoperative cardiac function associated with C34T mutation in donors, but worse 1-year survival. Our other studies on the metabolic impact of AMPD1 C34T mutation revealed decrease in AMPD activity, increased production of adenosine and de-inhibition of AMP regulated protein kinase. Thus, genetic, clinical and biochemical studies revealed that while long term attenuation of AMPD activity could be deleterious, transient inhibition of AMPD activity before acute cardiac injury is protective. We suggest therefore that pharmacological inhibition of AMP deaminase before transient ischemic event such as during ischemic heart disease or cardiac surgery could provide therapeutic benefit.

Chemerin gene expression is regulated by food restriction and food restriction-refeeding in rat adipose tissue but not in liver.

Chemerin is an adipokine that regulates adipocyte development and metabolism as well as inflammatory and immune function of some cells. Although chemerin may be linked to obesity and related diseases, little is known about the nutritional regulation of chemerin gene expression. We investigated the effect of prolonged food restriction, a common approach in treating obesity and related diseases, and prolonged food restriction-refeeding on chemerin gene expression in rat white adipose tissue and liver. The prolonged food restriction was accompanied by an approximately 2-fold decrease in chemerin mRNA level in rat white adipose tissue. Upon refeeding, an increase (approximately 8-fold as compared to rats maintained on restricted diet and 4-fold as compared to control) in chemerin mRNA level in white adipose tissue was found. Surprisingly, no effect of food restriction and food restriction-refeeding on chemerin mRNA level in the liver was found. Chemerin mRNA level in adipose tissue was positively correlated with serum insulin concentration. Moreover insulin increased significantly chemerin gene expression in primary rat adipocytes. The changes in chemerin mRNA level in adipose tissue and serum chemerin concentrations were associated with changes in serum leptin and free fatty acid concentrations. Collectively, the data presented here indicate that chemerin gene expression is regulated by nutritional status in rat adipose tissue but not in liver. It seems that insulin plays important role in stimulation of chemerin gene expression in adipose tissue. However, changes in serum leptin and free fatty acids concentrations after food restriction-refeeding suggest that the role of these factors in the regulation of chemerin gene expression in adipose tissue cannot be excluded. Lack of the effect of food restriction and food restriction-refeeding on liver chemerin gene expression suggests that adipose tissue is the dietary modifiable source of serum chemerin concentration.

Up-regulation of stearoyl-CoA desaturase 1 and elongase 6 genes expression in rat lipogenic tissues by chronic food restriction and chronic food restriction/refeeding.

Successful treatment of obesity and related diseases by chronic food restriction requires the understanding of the effect of such nutritional therapy on the expression of genes which have been implicated to be involved in some diseases associated with obesity. The purpose of this study was to examine the effect of chronic food restriction and chronic food restriction/refeeding on lipogenic enzymes, especially the expression of genes encoding the stearoyl-CoA desaturase 1 (Scd1) and elongase6 (Elovl6) in rat liver and adipose tissue. We found that both chronic food restriction and chronic food restriction/refeeding caused increased expression of the Scd1 and Elovl6 genes in both the liver and adipose tissue. The increase was more pronounced in case of chronic food restriction/refeeding (several-fold increase) than that in chronic food restriction alone (two to threefold increase). Essentially, similar results were obtained when the expression of fatty acid synthase, acetyl-CoA carboxylase, ATP-citrate lyase, and malic enzyme genes was studied. Moreover, we found that chronic food restriction and short-term fasting exert opposite effects on the expression of lipogenic enzymes genes. The increased expression of the genes encoding Scd1, Elovl6, and other key lipogenic enzymes may favor fat storage after chronic food restriction/refeeding and may be part of the molecular mechanism by which food restriction/refeeding increases body weight and enhances susceptibility to insulin resistance.

Differential effect of prolonged food restriction and fasting on hypothalamic malonyl-CoA concentration and expression of orexigenic and anorexigenic neuropeptides genes in rats.

Several lines of evidence suggest that malonyl-CoA in the hypothalamus plays an important role in monitoring and modulating body energy balance. In fasted state the level of malonyl-CoA concentration significantly decreases. Simultaneously, orexigenic neuropeptides (NPY - neuropeptide Y, AgRP - agouti-related peptide) genes are expressed at high level, whereas anorexigenic neuropeptides (CART - cocaine-and amphetamine-regulated transcript, POMC - proopiomelanocortin) genes are expressed at low level. When food intake resumes, opposite effect is observed. This study examined the effect of prolonged food restriction, common in humans trying to lose body weight on expression of orexigenic and anorexigenic neuropeptides genes and on malonyl-CoA content in rat whole hypothalamus. We observed an increase of NPY and AgRP mRNA levels in hypothalamus of rats kept on 30 days-long food restriction (50% of the amount of food consumed by controls). Simultaneously, a decrease of CART and POMC mRNA levels occurred. Refeeding caused a decrease in NPY and POMC mRNA levels without effect on AgRP and CART mRNA. Surprisingly, both prolonged food restriction and food restriction/refeeding caused the increase of malonyl-CoA level in whole hypothalamus. In contrast, fasting for 24h caused the decrease of malonyl-CoA level, which was associated with the up-regulation of NPY and AgRP genes expression and down-regulation of CART and POMC genes expression. After refeeding opposite effect was observed. These results indicate that prolonged food restriction and acute fasting, conditions in which energy expenditure exceeds intake, differentially affect malonyl-CoA concentration and similarly affect orexigenic and anorexigenic neuropeptide genes expression in whole rat hypothalamus.

Up-regulation of rat adipose tissue adiponectin gene expression by long-term but not by short-term food restriction.

Beneficial effects of food restriction as well as elevated circulating adiponectin concentration on the cardiovascular system, lipid metabolism and non-insulin dependent diabetes have been reported. The present article indicates that the reduction in rat body and adipose tissue weight after long-term (1 month) food restriction (either 75% or 50% of ad libitum food intake) was accompanied by the increase in serum adiponectin concentration. The increase in serum adiponectin concentration is the result of the significant increase in white adipose tissue adiponectin gene expression. In contrast to long-term food restriction, short-term (3 days) food restriction (either 75% or 50% of ad libitum food intake) did not influence body and fat mass as well as serum adiponectin concentration and white adipose tissue adiponectin mRNA level. The obtained results suggest that long-term food restriction is necessary to increase in serum adiponectin concentration that could be beneficial, to prevent some disorders associated with low serum adiponectin concentration like obesity and obesity-related diseases.

Genomic instability analysis of urine sediment versus tumor tissue in transitional cell carcinoma of the urinary bladder.

Microsatellite alterations are a common feature of neoplastic cells. Our study aimed to compare the profile of microsatellite DNA alterations in tumor tissue and urine sediment at 12 selected microsatellite loci in transitional cell carcinoma of the bladder, and to determine which of the 12 markers or combination of markers has potential for the non-invasive diagnosis of bladder cancer. DNA alterations were examined using microsatellite markers on chromosomes 2p, 3p, 8p, 9p, 9q, 12q, 13q, 17p and 18q in 38 patients, including 12 with superficial Ta/T1 and 26 with muscle invasive T2-T4 bladder tumors. Microsatellite instability was a rare event in comparison with loss of heterozygosity and was related to a low rate of defects in mismatch repair genes. The sensitivity of microsatellite analysis was 75% (9/12) for Ta/T1 tumors and 69% (18/26) for T2-T4 tumors. Two tetranucleotide markers, D9S242 and D9S252, when combined, displayed microsatellite alterations in 59% (16/27) of microsatellite analysis-positive patients. DNA alterations were not detected in 21 non-tumor specimens. Twenty of 51 (39%) tumor DNA alterations were re-detected in urine sediments, and 7 alterations found in urine sediments were not found in the corresponding tumor specimens. No association was found between the DNA alterations and any of the prognostic parameters. However, the overall survival correlated with microsatellite alterations (P=0.04, log-rank test). These data suggest that markers at tetranucleotide repeats on chromosome 9q have particular diagnostic potential in bladder cancer. Moreover, microsatellite analysis is suitable for the selection of patients with a less favorable outcome.

Enhanced citrate synthase activity in human pancreatic cancer.

OBJECTIVES: Assuming that a high flux of carbohydrate is strictly connected with lipid synthesis in neoplastic cells, one can hypothesize that the activity of citrate synthase, which plays an important role in glucose to lipid conversion, is enhanced in pancreatic cancer. The aim of the present study was to verify this hypothesis. METHODS: The activity of citrate synthase (as well as lactate and glucose 6-phosphate dehydrogenases) was measured using tissue extract prepared from specimens (pancreatic cancer and control specimens taken from the adjacent pancreatic normal tissue) obtained from 24 patients with ductal carcinoma who underwent pancreatoduodenectomy or total pancreatomy. RESULTS: The average of citrate synthase activity in human pancreatic ductal carcinoma is significantly higher comparing with adjacent nonneoplastic tissue: 40.2 +/- 27.2 and 18.3 +/- 13.6 nmole/min/mg protein, respectively (P = 0.001). The lactate dehydrogenase and glucose 6-phosphate dehydrogenase activity in human pancreatic ductal carcinoma were also higher than in adjacent nonneoplastic tissues. CONCLUSION: It is likely that enhanced citrate synthase activity contributes to the conversion of glucose to lipids in pancreatic cancer providing substrate for membrane lipids synthesis.