Metabolomic profiling of plasma from middle-aged and advanced-age male mice reveals the metabolic abnormalities of carnitine biosynthesis in metallothionein gene knockout mice.

Metallothionein (MT) is a family of low molecular weight, cysteine-rich proteins that regulate zinc homeostasis and have potential protective effects against oxidative stress and toxic metals. MT1 and MT2 gene knockout (MTKO) mice show shorter lifespans than wild-type (WT) mice. In this study, we aimed to investigate how MT gene deficiency accelerates aging. We performed comparative metabolomic analyses of plasma between MTKO and WT male mice at middle age (50-week-old) and advanced age (100-week-old) using liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS). The concentration of N6,N6,N6-trimethyl-L-lysine (TML), which is a metabolic intermediate in carnitine biosynthesis, was consistently higher in the plasma of MTKO mice compared to that of WT mice at middle and advanced age. Quantitative reverse transcription PCR (RT-PCR) analysis revealed remarkably lower mRNA levels of Tmlhe, which encodes TML dioxygenase, in the liver and kidney of male MTKO mice compared to that of WT mice. L-carnitine is essential for ß-oxidation of long-chain fatty acids in mitochondria, the activity of which is closely related to aging. Our results suggest that reduced carnitine biosynthesis capacity in MTKO mice compared to WT mice led to metabolic disorders of fatty acids in mitochondria in MTKO mice, which may have caused shortened lifespans.

Distinct kinetics for nucleotide hydrolysis in lymphocytes isolated from blood, spleen and cervical lymph nodes: Characterization of ectonucleotidase activity.

Ectonucleotidases are a plasma membrane-bound enzyme that hydrolyses extracellular adenosine triphosphate (eATP) and adenosine diphosphate (eADP) to adenosine monophosphate (AMP). It regulates normal function of lymphocytes, acts as an inflammatory marker and represents a molecular target for new therapeutics. Thus, this study sought to isolate lymphocytes from blood (BL), spleen (SL) and cervical lymph node (CLL), and characterize the eATP and eADP enzymatic hydrolysis in Wistar rats. The hydrolysis of the nucleotides occurred primarily at pH 8.0, 37°C in the presence of Ca2+ or Mg2+ . Chevillard-plot showed the hydrolysis of eATP and eADP at the same active site. The inhibitors of some classical ATDPases did not cause any significant change on enzymatic activity. Inhibitors of E-NTPDase (-1, -2, -3 isoforms) and E-NPP-1 decrease the enzyme activity in all resident lymphocytes. Furthermore, kinetic parameters (Vmax and Km) revealed that SL had significantly (P < .001) higher enzymatic activity when compared to BL and CLL. In conclusion, this study standardized kinetic values for eATP and eADP hydrolysis for resident lymphocytes isolated from BL, SL and CLL.

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.

Liver nucleotide biosynthesis is linked to protection from vascular complications in individuals with long-term type 1 diabetes.

Identification of biomarkers associated with protection from developing diabetic complications is a prerequisite for an effective prevention and treatment. The aim of the present study was to identify clinical and plasma metabolite markers associated with freedom from vascular complications in people with very long duration of type 1 diabetes (T1D). Individuals with T1D, who despite having longer than 30 years of diabetes duration never developed major macro- or microvascular complications (non-progressors; NP) were compared with those who developed vascular complications within 25 years from diabetes onset (rapid progressors; RP) in the Scandinavian PROLONG (n = 385) and DIALONG (n = 71) cohorts. The DIALONG study also included 75 healthy controls. Plasma metabolites were measured using gas and/or liquid chromatography coupled to mass spectrometry. Lower hepatic fatty liver indices were significant common feature characterized NPs in both studies. Higher insulin sensitivity and residual ß-cell function (C-peptide) were also associated with NPs in PROLONG. Protection from diabetic complications was associated with lower levels of the glycolytic metabolite pyruvate and APOCIII in PROLONG, and with lower levels of thiamine monophosphate and erythritol, a cofactor and intermediate product in the pentose phosphate pathway as well as higher phenylalanine, glycine and serine in DIALONG. Furthermore, T1D individuals showed elevated levels of picolinic acid as compared to the healthy individuals. The present findings suggest a potential beneficial shunting of glycolytic substrates towards the pentose phosphate and one carbon metabolism pathways to promote nucleotide biosynthesis in the liver. These processes might be linked to higher insulin sensitivity and lower liver fat content, and might represent a mechanism for protection from vascular complications in individuals with long-term T1D.

Plasma nucleotide reverse transcriptase inhibitor concentration and their associations with liver and renal parameters in people living with HIV.

: Associations between markers of liver and renal dysfunction and nucleotide reverse transcriptase inhibitor plasma exposure are ill-defined. As part of a large cohort study (Pharmacokinetic and Clinical Observations in People over Fifty), we analysed associations between alanine aminotransferase and estimated glomerular filtration rate results in people living with HIV on tenofovir disoproxil fumarate, emtricitabine, abacavir and lamivudine. While we found no associations between nucleotide reverse transcriptase inhibitor concentrations and alanine aminotransferase, lower estimated glomerular filtration rate values were associated with greater tenofovir, emtricitabine and lamivudine exposure, whereas abacavir showed no associations.

Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples.

We report a reverse phase chromatography mass spectrometry (LC-MS) method for simultaneous quantification of nucleosides and nucleotides from biological samples, where compound identification was achieved by a tier-wise approach and compound quantification was achieved via external calibration. A total of 65 authentic standards of nucleosides and nucleotides were used for the platform development. The limit of detection (LOD) of those compounds ranged from 0.05 nmol/L to 1.25 µmol/L, and their limit of quantification (LOQ) ranged from 0.10 nmol/L to 2.50 µmol/L. Using the developed method, nucleosides and nucleotides from human plasma, human urine, and rat liver were quantified. Seventy-nine nucleosides and nucleotides were identified from human urine and 28 of them were quantified with concentrations of 13.0 nmol/L-151 µmol/L. Fifty-five nucleosides and nucleotides were identified from human plasma and 22 of them were quantified with concentrations of 1.21 nmol/L-8.54 µmol/L. Fifty-one nucleosides and nucleotides were identified from rat liver and 23 were quantified with concentrations of 1.03 nmol/L-31.7 µmol/L. These results demonstrate that the developed method can be used to investigate the concentration change of nucleosides and nucleotides in biological samples for the purposes of biomarker discovery or elucidation of disease mechanisms.

Extraction of nucleobases, nucleosides and nucleotides by employing a magnetized graphene oxide functionalized with hydrophilic phytic acid and titanium(IV) ions.

A magnetite@graphene oxide nanocomposite was first coated with polyethylenimine and then modified with phytic acid and titanium(IV) ions. The high loading with Ti(IV) and the good hydrophilicity of PEI and PA result in a material that can be applied to the efficient extraction of highly polar nucleobases, nucleosides and nucleotides. The physicochemical properties of the composite were investigated by scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, water contact angle measurements, thermogravimetric analysis, and vibrating sample magnetometry. A series of parameters that affect extraction and elution under the conditions of immobilized metal affinity chromatography (IMAC) and hydrophilic interaction liquid chromatography (HILIC) were examined. The analytes were eluted from the nanocomposites using 10 mM trisodium phosphate as the elution solution in the IMAC mode, and 50% methanol-water as elution solution in the HILIC mode. Figures of merit include (a) an intra-day precision of 0.1-1.0% in the IMAC mode; (b) an intra-day precision of 0.4%-0.8% in the HILIC mode; (c) detection limits between 1.8-2.8 ng mL-1 in the IMAC mode; and (d) detection limits of 4.0-10.5 ng mL-1 in the HILIC mode. The method was applied to the extraction of the nucleotides cytidine-5'-monophosphate (CMP), uridine-5'-monophosphate (UMP), guanosine-5'-monophosphate (GMP), and adenosine-5'-monophosphate (AMP), and the nucleobases and nucleosides hypoxanthine, adenosine, cytosine, inosine and cytidine from Cordyceps sinensis, Lentinus edodes and plasma samples. Graphical abstract Schematic presentation of the workflow for the extraction of nucleobases, nucleosides and nucleotides using phytic acid-Ti(IV) functionalized magnetite@graphene oxide nanocomposites under two distinct modes.

Quantification of Thiopurine Nucleotides in Erythrocytes and Clinical Application to Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: Concentrations of 6-thioguanine (6TG) nucleotides and 6-methylmercaptopurine (6MMP) nucleotides in RBCs were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay was validated for clinical use and was applied to blood samples from patients taking mercaptopurine (6MP). METHODS: RBCs were hemolyzed and deproteinized using perchloric acid, followed by heating for the hydrolysis of nucleotides, and the resultant base was measured using LC-MS/MS. Precision, recovery, linearity, matrix effect, and limit of quantification was validated for clinical application. Our results were compared with another institution's established LC-MS/MS assay. We measured the concentrations of 6TG and 6MMP in RBCs of pediatric patients with acute lymphoblastic leukemia (ALL), and the clinical impact of those metabolites was investigated. RESULTS: The imprecision coefficient of variations of 6TG and 6MMP were 5.7%-8.1%, and the bias was within 5%. Lower limits of quantification were set at 54 ng/mL for 6TG and 1036 ng/mL for 6MMP. Correlation coefficients for 6TG and 6MMP were 0.997 and 1.0 in a comparison study. For clinical proof-of-concept, 74 blood samples were collected from 37 pediatric ALL patients receiving maintenance therapy. Concentration of 6TG ranged from 16.1 to 880 pmol/8 × 10 RBCs and that of 6MMP from 55 to 20,937 pmol/8 × 10 RBCs. The 6MP metabolites were not correlated with WBC or absolute neutrophil count. On the other hand, the higher 6MMP level was associated with elevated alanine aminotransferase and aspartate aminotransferase. CONCLUSIONS: In this study, an assay for the quantification of 6TG and 6MMP in RBCs was established and applied to pediatric ALL patients. Interindividual variability in 6MP metabolite concentrations was considerable and associated with elevation of liver enzymes, which may be useful in the clinical monitoring of 6MP maintenance therapy in pediatric ALL patients.

Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices.

Nucleotides play a role in inflammation processes: cAMP and cGMP in the endothelial barrier function, ADP in platelet aggregation, ATP and UTP in vasodilatation and/or vasoconstriction of blood vessels, UDP in macrophages activation. The aim of this study is to develop and validate a LC/MS-MS method able to quantify simultaneously nine nucleotides (AMP, cAMP, ADP, ATP, GMP, cGMP, UMP, UDP and UTP) in biological matrixes (cells and plasma). The method we developed, has lower LOQ's than others and has the main advantage to quantify all nucleotides within one single injection in less than 10 min. The measured nucleotides concentrations obtained with this method are similar to those obtained with assay kits commercially available. Analysis of plasma and red blood cells from healthy donors permits to estimate the physiological concentration of those nucleotides in human plasma and red blood cells, such information being poorly available in the literature. Furthermore, the protocol presented in this paper allowed us to observe that AMP, ADP, ATP concentrations are modified in human red blood cells and plasma after a venous stasis of 4 min compared to physiological blood circulation. Therefore, this specific method enables future studies on nucleotides implications in chronic inflammatory diseases but also in other pathologies where nucleotides are implicated in.

Plasma Nucleotide Dynamics during Exercise and Recovery in Highly Trained Athletes and Recreationally Active Individuals.

Circulating plasma ATP is able to regulate local skeletal muscle blood flow and 02 delivery causing considerable vasodilatation during exercise. We hypothesized that sport specialization and specific long-term training stimuli have an impact on venous plasma [ATP] and other nucleotides concentration. Four athletic groups consisting of sprinters (n=11; age range 21-30 yr), endurance-trained athletes (n=16; age range 18-31 yr), futsal players (n=14; age range 18-30 yr), and recreationally active individuals (n=12; age range 22-33 yr) were studied. Venous blood samples were collected at rest, during an incremental treadmill test, and during recovery. Baseline [ATP] was 759±80 nmol·l-1 in competitive athletes and 680±73 nmol·l-1 in controls and increased during exercise by ~61% in competitive athletes and by ~31% in recreationally active participants. We demonstrated a rapid increase in plasma [ATP] at exercise intensities of 83-87% of VO2max in competitive athletes and 94% in controls. Concentrations reported after 30 minutes of recovery were distinct from those obtained preexercise in competitive athletes (P < 0.001) but not in controls (P = 0.61). We found a correlation between total-body skeletal muscle mass and resting and maximal plasma [ATP] in competitive athletes (r=0.81 and r=0.75, respectively). In conclusion, sport specialization is significantly related to plasma [ATP] at rest, during exercise, and during maximal effort. Intensified exercise-induced plasma [ATP] increases may contribute to more effective vessel dilatation during exercise in highly trained athletes than in recreational runners. The most rapid increase in ATP concentration was associated with the respiratory compensation point. No differences between groups of competitive athletes were observed during the recovery period suggesting a similar pattern of response after exercise. Total-body skeletal muscle mass is indirectly related to plasma [ATP] in highly trained athletes.

Adenosine deaminase inhibition suppresses progression of 4T1 murine breast cancer by adenosine receptor-dependent mechanisms.

The activity of a cell-surface ecto-adenosine deaminase (eADA) is markedly increased in the endothelial activation and vascular inflammation leading to decreased adenosine concentration and alterations in adenosine signalling. Depending on the specific pathway activated, extracellular purines mediate host cell response or regulate growth and cytotoxicity on tumour cells. The aim of this study was to test the effects of adenosine deaminase inhibition by 2'deoxycoformycin (dCF) on the breast cancer development. dCF treatment decreased a tumour growth and a final tumour mass in female BALB/c mice injected orthotopically with 4T1 cancer cells. dCF also counteracted cancer-induced endothelial dysfunction in orthotopic and intravenous 4T1 mouse breast cancer models. In turn, this low dCF dose had a minor effect on immune stimulation exerted by 4T1 cell implantation. In vitro studies revealed that dCF suppressed migration and invasion of 4T1 cells via A2a and A3 adenosine receptor activation as well as 4T1 cell adhesion and transmigration through the endothelial cell layer via A2a receptor stimulation. Similar effects of dCF were observed in human breast cancer cells. Moreover, dCF improved a barrier function of endothelial cells decreasing its permeability. This study highlights beneficial effects of adenosine deaminase inhibition on breast cancer development. The inhibition of adenosine deaminase activity by dCF reduced tumour size that was closely related to the decreased aggressiveness of tumour cells by adenosine receptor-dependent mechanisms and endothelial protection.

Reproducibility of non-fasting plasma metabolomics measurements across processing delays.

INTRODUCTION: Processing delays after blood collection is a common pre-analytical condition in large epidemiologic studies. It is critical to evaluate the suitability of blood samples with processing delays for metabolomics analysis as it is a potential source of variation that could attenuate associations between metabolites and disease outcomes. OBJECTIVES: We aimed to evaluate the reproducibility of metabolites over extended processing delays up to 48 h. We also aimed to test the reproducibility of the metabolomics platform. METHODS: Blood samples were collected from 18 healthy volunteers. Blood was stored in the refrigerator and processed for plasma at 0, 15, 30, and 48 h after collection. Plasma samples were metabolically profiled using an untargeted, ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) platform. Reproducibility of 1012 metabolites over processing delays and reproducibility of the platform were determined by intraclass correlation coefficients (ICCs) with variance components estimated from mixed-effects models. RESULTS: The majority of metabolites (approximately 70% of 1012) were highly reproducible (ICCs ≥ 0.75) over 15-, 30- or 48-h processing delays. Nucleotides, energy-related metabolites, peptides, and carbohydrates were most affected by processing delays. The platform was highly reproducible with a median technical ICC of 0.84 (interquartile range 0.68-0.93). CONCLUSION: Most metabolites measured by the UPLC-MS/MS platform show acceptable reproducibility up to 48-h processing delays. Metabolites of certain pathways need to be interpreted cautiously in relation to outcomes in epidemiologic studies with prolonged processing delays.

A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells.

PURPOSE: To develop and validate a method for the simultaneous measurement of adenosine, guanosine, and inosine derived from mono (MP) and triphosphate (TP) forms in peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs) and dried blood spots (DBS). METHODS: Solid phase extraction of cell lysates followed by dephosphorylation to molar equivalent nucleoside and LC-MS/MS quantification. RESULTS: The assay was linear for each of the three quantification ranges: 10-2000, 1.0-200 and 0.25-50 pmol/sample for adenosine, guanosine, and inosine, respectively. Intraassay (n = 6) and interassay (n = 18) precision (%CV) were within 1.7 to 16% while accuracy (%deviation) was within -11.5 to 14.7% for all three analytes. Nucleotide monophosphates were less concentrated than triphosphates (except for inosine) and levels in PBMCs were higher than RBCs for all three nucleotides (10, 55, and 5.6 fold for ATP, GTP and ITP, respectively). DBS samples had an average (SD) of -26% (22.6%) lower TP and 184% (173%) higher MP levels compared to paired RBC lysates, suggesting hydrolysis of the TP in DBS. CONCLUSION: This method was accurate and precise for physiologically relevant concentrations of adenosine, guanosine and inosine nucleotides in mono- and triphosphate forms, providing a bioanalytical tool for quantitation of nucleotides for clinical studies.

Effects of ribavirin/sofosbuvir treatment and ITPA phenotype on endogenous purines.

Ribavirin (RBV), a purine analog, causes hemolytic anemia in some patients. In vitro, anemia appears to result from depletion of endogenous purines, but there are limited data in vivo. Single nucleotide polymorphisms in the gene encoding the inosine triphosphatase (ITPA) enzyme have been associated with protection against RBV-induced anemia and may mediate the effect of RBV treatment on endogenous purines. The purpose of this work was to determine the effect of RBV treatment on endogenous purine concentrations in individuals being treated for chronic hepatitis C virus (HCV) infection. Adenosine triphosphate (ATP), guanosine triphosphate (GTP), inosine triphosphate (ITP) and ribavirin triphosphate (RTP) were measured in whole blood obtained from 47 HCV-infected individuals at day zero (baseline), day three, day 28 and day 84 of RBV/sofosbuvir (SOF) treatment. ATP decreased -35.1% and -38.6% (p < 0.0001) at day 28 and day 84 of treatment, respectively compared to baseline. The decrease in ATP was greater in patients with ≤60% ITPA activity compared to those with 100% ITPA activity (-29.4% vs. -9.6%). GTP did not change during treatment but was 16.5% (p = 0.01) higher per 100 pmol/106 cells RTP in those with 100% ITPA activity. No significant change or effect of RTP or ITPA phenotype was noted for ITP. In summary, only ATP was reduced by RBV/SOF treatment and ITPA variants had larger reductions in ATP suggesting RBV-induced anemia is due to a different mechanism than predicted from in-vitro studies. These data emphasize the importance of characterizing the effect of nucleos(t)ide analog treatment on endogenous purines in-vivo.

Tenofovir/emtricitabine metabolites and endogenous nucleotide exposures are associated with p16(INK4a) expression in subjects on combination therapy.

BACKGROUND: HIV may amplify immunological, physiological and functional changes of ageing. We determined associations of frailty phenotype, a T-cell senescence marker (p16(INK4a) expression), age and demographics with exposures of the intracellular metabolites (IM) and endogenous nucleotides (EN) of tenofovir/emtricitabine (TFV/FTC), efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV). METHODS: Plasma and peripheral blood mononuclear cell samples for drug, IM and EN concentrations were collected at four time points in HIV+ adults receiving TFV/FTC with EFV or ATV/RTV. Subjects underwent frailty phenotyping and p16(INK4a) expression analysis. Non-compartmental analysis generated an area under the curve (AUC) for each analyte. Spearman rank correlation and Kruskal-Wallis tests were used to assess associations between AUC, demographics and ageing markers, adjusting for multiple comparisons with the Holm procedure. RESULTS: Subjects (n=79) ranged in age from 22-73 years (median 48 years); 48 were African-American, 24 were female, 54 received EFV. Three subjects (range 51-60 years) demonstrated frailty, with 17 subjects (range 26-60 years) demonstrating pre-frailty. Negative associations were observed between p16(INK4a) expression and each of FTC-triphosphate (r=-0.45), deoxyadenosine triphosphate (dATP; r=-0.47) and deoxycytidine triphosphate (dCTP; r=-0.57) AUCs (P-values <0.02). TFV and FTC AUCs were larger among subjects with lower renal function or higher chronological age (P-values ≤0.05). No associations were observed for EFV, ATV or RTV AUCs. CONCLUSIONS: Associations of IM/EN exposure and p16(INK4a) expression observed here suggest that senescence may alter drug phosphorylation, metabolism or transport. This finding warrants further mechanistic study to ensure optimal treatment in the ageing HIV+ population. Clinicaltrials.gov NCT01180075.

Expression of microRNAs in Horse Plasma and Their Characteristic Nucleotide Composition.

MicroRNAs (miRNAs) in blood plasma are stable under high levels of ribonuclease activity and could function in tissue-to-tissue communication, suggesting that they may have distinctive structural characteristics compared with non-circulating miRNAs. In this study, the expression of miRNAs in horse plasma and their characteristic nucleotide composition were examined and compared with non-plasma miRNAs. Highly expressed plasma miRNA species were not part of the abundant group of miRNAs in non-plasma tissues, except for the eca-let-7 family. eca-miR-486-5p, -92a, and -21 were among the most abundant plasma miRNAs, and their human orthologs also belong to the most abundant group of miRNAs in human plasma. Uracil and guanine were the most common nucleotides of both plasma and non-plasma miRNAs. Cytosine was the least common in plasma and non-plasma miRNAs, although levels were higher in plasma miRNAs. Plasma miRNAs also showed higher expression levels of miRNAs containing adenine and cytosine repeats, compared with non-plasma miRNAs. These observations indicate that miRNAs in the plasma have a unique nucleotide composition.

Metabolic Characterization of the Common Marmoset (Callithrix jacchus).

High-resolution metabolomics has created opportunity to integrate nutrition and metabolism into genetic studies to improve understanding of the diverse radiation of primate species. At present, however, there is very little information to help guide experimental design for study of wild populations. In a previous non-targeted metabolomics study of common marmosets (Callithrix jacchus), Rhesus macaques, humans, and four non-primate mammalian species, we found that essential amino acids (AA) and other central metabolites had interspecies variation similar to intraspecies variation while non-essential AA, environmental chemicals and catabolic waste products had greater interspecies variation. The present study was designed to test whether 55 plasma metabolites, including both nutritionally essential and non-essential metabolites and catabolic products, differ in concentration in common marmosets and humans. Significant differences were present for more than half of the metabolites analyzed and included AA, vitamins and central lipid metabolites, as well as for catabolic products of AA, nucleotides, energy metabolism and heme. Three environmental chemicals were present at low nanomolar concentrations but did not differ between species. Sex and age differences in marmosets were present for AA and nucleotide metabolism and warrant additional study. Overall, the results suggest that quantitative, targeted metabolomics can provide a useful complement to non-targeted metabolomics for studies of diet and environment interactions in primate evolution.

Liquid chromatography/tandem mass spectrometry method for simultaneous quantification of eight endogenous nucleotides and the intracellular gemcitabine metabolite dFdCTP in human peripheral blood mononuclear cells.

Quantification of endogenous nucleotides is of interest for investigation of numerous cellular biochemical processes, such as energy metabolism and signal transduction, and may also be applied in cancer and antiretroviral therapies in which nucleoside analogues are used. For these purposes we developed and validated a sensitive and high accuracy ion-pair liquid chromatography tandem mass spectrometry (IP LC-MS/MS) method for simultaneous quantification of eight endogenous nucleotides (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, dTTP) and 2',2'-difluoro-2'-deoxycytidine triphosphate (dFdCTP), an intracellular metabolite of the nucleoside analogue gemcitabine. The assay was validated using 200µL aliquots of peripheral blood mononuclear cell (20×10(6)cells/ml, 4×10(6)cells) extracts, pretreated with activated charcoal and spiked with unlabeled nucleotides, deoxynucleotides and dFdCTP. Analytes were extracted by simple precipitation with cold 60% methanol containing isotope labeled internal standards and separated on a porous graphitic carbon column. For method validation, the concentration ranges were: 0.125-20.8pmol injected for deoxynucleotides, 0.25-312.5pmol injected for dFdCTP and 5-3200pmol injected for nucleotides. The highest coefficients of variation (CV) were 12.1% for within run assay and 11.4% for between run assay, both representing the precision at the lowest analyte concentrations. The method was applied to monitor dFdCTP and changes in endogenous nucleotides in patients who were receiving gemcitabine infusions.

Role of Endogenous Factors in Response of Erythrocyte Membrane in Patients with Cardiovascular Diseases under Conditions of Ischemic Exposure.

We studied specific features of erythrocyte membrane response to short-term occlusion of the brachial artery in patients with cardiovascular pathology. Under ischemic conditions, processes of sorption were primarily intensified in patients with effort angina and processes of hemoglobin binding with erythrocyte membrane predominated in patients with essential hypertension. These changes in the cell membrane were related to modulation of aggregation properties of erythrocytes (in patients with angina) and plasminogen activity (in patients with essential hypertension). They can also be associated with changes in glucose levels (effort angina) and uric acid (essential hypertension) whose effects can be significantly modified by other endogenous factors.

Blood oxygen affinity increases during digestion in the South American rattlesnake, Crotalus durissus terrificus.

Digesting snakes experience massive increases in metabolism that can last for many days and are accompanied by adjustments in the oxygen transport cascade. Accordingly, we examined the oxygen-binding properties of the blood in the South American rattlesnake (Crotalus durissus terrificus) during fasting and 24 and 48h after the snakes have ingested a rodent meal corresponding to 15% (±2%) of its own body mass. In general, oxygen-hemoglobin (Hb-O2) affinity was significantly increased 24h post-feeding, and then returned toward fasting values within 48h post-feeding. Content of organic phosphates ([NTP] and [NTP]/[Hb]), hemoglobin cooperativity (Hill's n), and Bohr Effect (ΔlogP50/ΔpH) were not affected by feeding. The postprandial increase in Hb-O2 affinity in the South American rattlesnake can be almost entirely ascribed by the moderate alkaline tide that follows meal ingestion. In general, digesting snakes were able to regulate blood metabolites at quite constant levels (e.g., plasma osmolality, lactate, glucose, and total protein levels). The level of circulating lipids, however, was considerably increased, which may be related to their mobilization, since lipids are known to be incorporated by the enterocytes after snakes have fed. In conclusion, our results indicate that the exceptional metabolic increment exhibited by C. d. terrificus during meal digestion is entirely supported by the aerobic pathways and that among the attending cardiorespiratory adjustments, pulmonary Hb-O2 loading is likely improved due to the increment in blood O2 affinity.