Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells.

RESEARCH QUESTION: Female age is the single greatest factor influencing reproductive performance and granulosa cells are considered as potential biomarkers of oocyte quality. Is there an age effect on the energy metabolism of human mural granulosa cells? DESIGN: Observational prospective cohort and experimental study including 127 women who had undergone IVF cycles. Women were allocated to two groups: a group of infertile patients aged over 38 years and a control group comprising oocyte donors aged less than 35 years. Individuals with pathologies that could impair fertility were excluded from both groups. Following oocyte retrieval, cumulus and granulosa cells were isolated and their bioenergetic properties (oxidative phosphorylation parameters, rate of aerobic glycolysis and adenine nucleotide concentrations) were analysed and compared. RESULTS: Human mural luteinized granulosa and cumulus cells present high rates of aerobic glycolysis that cannot be increased further when mitochondrial ATP synthesis is inhibited. Addition of follicular fluid to the experimental media is necessary to reach the full respiratory capacity of the cells. Granulosa cells from aged women present lower mitochondrial respiration (12.8 ± 1.6 versus 11.2 ± 1.6 pmol O2/min/mg; P = 0.046), although mitochondrial mass is not decreased, and lower aerobic glycolysis, than those from young donors (12.9 ± 1.3 versus 10.9 ± 0.5 mpH/min/mg; P = 0.009). The concurrent decrease in the two energy supply pathways leads to a decrease in the cellular energy charge (0.87 ± 0.01 versus 0.83 ± 0.02; P < 0.001). CONCLUSIONS: Human mural luteinized granulosa cells exhibit a reduction in their energy metabolism as women age that is likely to influence female reproductive potential.

Colorimetric discrimination of nucleoside phosphates based on catalytic signal amplification strategy and its application to related enzyme assays.

Selective detection of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) which are less charged molecules than adenosine triphosphate (ATP) or pyrophosphate (PPi) in aqueous solution has been considered challenging because AMP and ADP have relatively low binding affinity for phosphate receptors. In this study, colorimetric discrimination of nucleoside phosphates was achieved based on catalytic signal amplification through the activation of artificial peroxidase. This method showed high selectivity for AMP and ADP over ATP and PPi, unlike previous phosphate sensors that use Zn2+-dipicolylamine-based receptors. High selectivity of the suggested method allowed discrimination of AMP in aqueous solution by the naked eye, and the detection limit was estimated to be 0.5 µM. Mechanism analysis revealed AMP acted as activators in the peroxidation cycle of the Mn2(bpmp)/ABTS/H2O2 system despite having relatively low binding affinity. Additionally, high selectivity and quantitative signal amplification allowed for the development of colorimetric phosphodiesterase and a small molecule kinase assay method. The newly proposed method offers direct, real-time, and quantitative analysis of enzyme activities and inhibition, and is expected to be further applied to high-throughput screening of inhibitors.

Supramolecular Assembly of Acriflavine on Graphene Oxide for the Sensing of Adenosine Phosphates.

An acriflavine-graphene oxide (GAF) supramolecular assembly has been prepared from water-soluble graphene oxide (GO) and a fluorescent dye, acriflavine (AF). Upon binding this non-covalently to the GO, the fluorescence of acriflavine has been "turned off" effectively, competitive binding potential of the sensor substrates such as ATP, ADP, AMP and the pyrophosphate weakens the supramolecular assembly of GAF, which allows the release of acriflavine quantitatively, which also "turns-on" the fluorescence of the dye under UV irradiation. Interestingly, GAF displayed the highest sensitivity towards ATP within the family of adenosine phosphates. We have developed a naked eye detection method for the adenosine phosphates biomolecules. For the first time, acriflavine has been utilized for the sensing of adenosine phosphates in combination with GO, which can be useful for the detection of other biomolecules.

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct rodent hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24 hour fasting period that resulted in substantially reduced muscle carbohydrate supply. 180 mins of acute hypoxia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product-inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Fasting and the associated reduction of the available intracellular carbohydrate pool (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics.

Xanthine oxidase inhibitor ameliorates postischemic renal injury in mice by promoting resynthesis of adenine nucleotides.

Although oxidative stress plays central roles in postischemic renal injury, region-specific alterations in energy and redox metabolism caused by short-duration ischemia remain unknown. Imaging mass spectrometry enabled us to reveal spatial heterogeneity of energy and redox metabolites in the postischemic murine kidney. After 10-minute ischemia and 24-hour reperfusion (10mIR), in the cortex and outer stripes of the outer medulla, ATP substantially decreased, but not in the inner stripes of the outer medulla and inner medulla. 10mIR caused renal injury with elevation of fractional excretion of sodium, although histological damage by oxidative stress was limited. Ischemia-induced NADH elevation in the cortex indicated prolonged production of reactive oxygen species by xanthine oxidase (XOD). However, consumption of reduced glutathione after reperfusion suggested the amelioration of oxidative stress. An XOD inhibitor, febuxostat, which blocks the degradation pathway of adenine nucleotides, promoted ATP recovery and exerted renoprotective effects in the postischemic kidney. Because effects of febuxostat were canceled by silencing of the hypoxanthine phosphoribosyl transferase 1 gene in cultured tubular cells, mechanisms for the renoprotective effects appear to involve the purine salvage pathway, which uses hypoxanthine to resynthesize adenine nucleotides, including ATP. These findings suggest a novel therapeutic approach for acute ischemia/reperfusion renal injury with febuxostat through salvaging high-energy adenine nucleotides.

Extending the Scope of 1H NMR Spectroscopy for the Analysis of Cellular Coenzyme A and Acetyl Coenzyme A.

Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) are ubiquitous cellular molecules, which mediate hundreds of anabolic and catabolic reactions including energy metabolism. Highly sensitive methods including absorption spectroscopy and mass spectrometry enable their analysis, albeit with many limitations. To date, however, NMR spectroscopy has not been used to analyze these important molecules. Building on our recent efforts, which enabled simultaneous analysis of a large number of metabolites in tissue and blood including many coenzymes and antioxidants ( Anal. Chem. 2016, 88, 4817-24; ibid 2017, 89, 4620-4627), we describe here a new method for identification and quantitation of CoA and acetyl-CoA ex vivo in tissue. Using mouse heart, kidney, liver, brain, and skeletal tissue, we show that a simple 1H NMR experiment can simultaneously measure these molecules. Identification of the two species involved a comprehensive analysis of the different tissue types using 1D and 2D NMR, in combination with spectral databases for standards, as well as spiking with authentic compounds. Time dependent studies showed that while the acetyl-CoA levels remain unaltered, CoA levels diminish by more than 50% within 24 h, which indicates that CoA is labile in solution; however, degassing the sample with helium gas halted its oxidation. Further, interestingly, we also identified endogenous coenzyme A glutathione disulfide (CoA-S-S-G) in tissue for the first time by NMR and show that CoA, when oxidized in tissue extract, also forms the same disulfide metabolite. The ability to simultaneously visualize absolute concentrations of CoA, acetyl-CoA, and endogenous CoA-S-S-G along with redox coenzymes (NAD+, NADH, NADP+, NADPH), energy coenzymes (ATP, ADP, AMP), antioxidants (GSH, GSSG), and a vast pool of other metabolites using a single 1D NMR spectrum offers a new avenue in the metabolomics field for investigation of cellular function in health and disease.

2D-Metal-Organic-Framework-Nanozyme Sensor Arrays for Probing Phosphates and Their Enzymatic Hydrolysis.

The detection of phosphates and their enzymatic hydrolysis is of great importance because of their essential roles in various biological processes and numerous diseases. Compared with individual sensors for detecting one given phosphate at a time, sensor arrays are able to discriminate multiple phosphates simultaneously. Although nanomaterial-based sensor arrays have shown great promise for the discrimination of phosphates, very few of them have been explored for probing phosphates involved enzymatic hydrolysis. To fill this gap, herein we fabricated two-dimensional-metal-organic-framework (2D-MOF)-nanozyme-based sensor arrays by modulating their peroxidase-mimicking activity with various phosphates, including AMP, ADP, ATP, pyrophosphate (PPi), and phosphate (Pi). The sensor arrays were used to successfully discriminate the five phosphates not only in aqueous solutions but also in biological samples. The practical application of the sensor arrays was then validated with blind samples, where 30 unknown samples containing phosphates were accurately identified. Moreover, the sensor arrays were successfully applied to probing hydrolytic processes involving ATP and PPi that are catalyzed by apyrase and PPase, respectively. This work demonstrates a nanozyme-based sensor array as a convenient and reliable analytical platform for probing phosphates and their related enzymatic processes, which could be applied to other analytes and enzymatic reactions.

Development and validation of a HILIC-UV method for the determination of nucleotides in fish samples.

The aim of this work was the development of a simple, novel and accurate method for the determination of adenosine triphosphate (ATP) and its first five catabolites: adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine (Ino) and hypoxanthine (Hx), in fish tissue, based on hydrophilic interaction liquid chromatography (HILIC). For this purpose, a stationary phase for polar and hydrophilic compounds (ZIC-pHILIC) was used. The effect of different chromatographic parameters and the molecular mechanism based on the van't Hoff plot were examined. The t-test and Dixon's Q-test were applied in order to examine statistical differences and outlier values. The recovery of the method ranged between 82.7% and 127% and the %RSD values were lower than 10% for all analytes determined. The method was applied in frozen sea bream samples stored at 0-4 °C. The Ki-, G-, H- and F values were calculated for the estimation of the level of fish freshness.

A sensitive method for the separation and quantification of low-level adenine nucleotides using porous graphitic carbon-based liquid chromatography and tandem mass spectrometry.

A liquid chromatography coupled to heated electrospray ionization/tandem mass spectrometry (LC-HESI-MS/MS) method was developed for the simultaneous quantitative analysis of low nanomolar level adenine nucleotides AMP, ADP, ATP, cyclic AMP (cAMP), and the nucleoside adenosine. For analyte retention and separation, reverse phase chromatography using porous graphitic carbon (PGC) was employed as it provided full resolution. The erratic chromatographic behaviour characteristic of PGC, including deterioration of analyte resolution and increased peak tailing (leading to decreased sensitivity), was mitigated by incorporating acidic equilibration within runs using a quaternary gradient. Analyte resolution and chromatographic sensitivity were still lost after a period of column inactivity; hence a pre-conditioning protocol was implemented between batches to regenerate the column. These column regeneration measures also allowed elution of AMP, ADP and ATP in the sequence of mono- to tri- nucleotides, differing from conventional reverse phase elution where analytes elute with decreasing polarity. This nucleotide elution sequence has the advantage of overcoming potential mis-annotation and inaccurate quantification of smaller nucleotides caused by in-source fragmentation of ATP. The method was validated in granulosa cell conditioned media, with the LLOQs ranging between 10-50nM for most analytes. To verify the method using biological samples, nucleotide secretion was measured in granulosa cell conditioned media under various treatments known to alter their levels. Moreover, the method was applied to cumulus-oocyte complex cell lysates to examine its linearity in a complex matrix.

Effects of dietary starch types on early postmortem muscle energy metabolism in finishing pigs.

This study aimed to investigate the effects of different dietary starch types on early postmortem muscle energy metabolism in finishing pigs. Ninety barrows (68.0±2.0kg) were randomly allotted to three experimental diets with five replicates of six pigs, containing pure waxy maize starch (WMS), nonwaxy maize starch (NMS), and pea starch (PS) (amylose/amylopectin were 0.07, 0.19 and 0.28 respectively). Compared with the WMS diet, pigs fed the PS diet exhibited greater creatine kinase activity, higher adenosine triphosphate and adenosine diphosphate contents, lower phosphocreatine (PCr), adenosine monophosphate and glycogen contents, and lower glycolytic potential (P<0.05). Moreover, the PS diet led to reduced percentage of bound hexokinase activity, decreased level of phosphorylated AKT (P<0.05) and increased level of hypoxia-inducible factor-1α (P<0.05). In conclusion, diet with high amylose content might promote PCr degradation and inhibit the rate of glycolysis, followed by attenuation of early postmortem glycolysis in finishing pigs.

Determination and quantification of intracellular fludarabine triphosphate, cladribine triphosphate and clofarabine triphosphate by LC-MS/MS in human cancer cells.

Purine nucleoside analogues are widely used in the treatment of haematological malignancies, and their biological activity is dependent on the intracellular accumulation of their triphosphorylated metabolites. In this context, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to study the formation of 5'-triphosphorylated derivatives of cladribine, fludarabine, clofarabine and 2'-deoxyadenosine in human cancer cells. Br-ATP was used as internal standard. Separation was achieved on a hypercarb column. Analytes were eluted with a mixture of hexylamine (5 mM), DEA (0.4%, v/v, pH 10.5) and acetonitrile, in a gradient mode at a flow rate of 0.3mLmin-1. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI-) were used for detection. The application of this method to the quantification of these phosphorylated cytotoxic compounds in a human follicular lymphoma cell line, showed that it was suitable for the study of relevant biological samples.

Contribution of mitochondria to injury of hepatocytes and liver tissue by hyperthermia.

OBJECTIVE: The aim of this study was to investigate functional changes of liver mitochondria within the experimentally modeled transition zone of radiofrequency ablation and to estimate possible contribution of these changes to the energy status of liver cells and the whole tissue. MATERIALS AND METHODS: Experiments were carried out on mitochondria isolated from the perfused liver and isolated hepatocytes of male Wistar rats. Hyperthermia was induced by changing the temperature of perfusion medium in the range characteristic for the transition zone (38-52°C). After 15-min perfusion, mitochondria were isolated to investigate changes in the respiration rates and the membrane potential. Adenine nucleotides extracted from isolated hepatocytes and perfused liver subjected to hyperthermic treatment were analyzed by HPLC. RESULTS: Hyperthermic liver perfusion at 42-52°C progressively impaired oxidative phosphorylation in isolated mitochondria. Significant inhibition of the respiratory chain components was observed after perfusion at 42°C, irreversible uncoupling became evident after liver perfusion at higher temperatures (46°C and above). After perfusion at 50-52°C energy supplying function of mitochondria was entirely compromised, and mitochondria turned to energy consumers. Hyperthermia-induced changes in mitochondrial function correlated well with changes in the energy status and viability of isolated hepatocytes, but not with the changes in the energy status of the whole liver tissue. CONCLUSIONS: In this study the pattern of the adverse changes in mitochondrial functions that are progressing with increase in liver perfusion temperature was established. Results of experiments on isolated mitochondria and isolated hepatocytes indicate that hyperthermic treatment significantly and irreversibly inhibits energy-supplying function of mitochondria under conditions similar to those existing in the radiofrequency ablation transition zone and these changes can lead to death of hepatocytes. However, it was not possible to estimate contribution of mitochondrial injury to liver tissue energy status by estimating only hyperthermia-induced changes in adenine nucleotide amounts on the whole tissue level.

Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid glycoprotein. Spectroscopic and molecular docking approach.

The binding interaction between clofarabine, an important anticancer drug and two important carrier proteins found abundantly in human plasma, Human Serum Albumin (HSA) and α-1 acid glycoprotein (AAG) was investigated by spectroscopic and molecular modeling methods. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of HSA and AAG is quenched by clofarabine and the static mode of fluorescence quenching is operative. UV-vis spectroscopy deciphered the formation of ground state complex between anticancer drug and the two studied proteins. Clofarabine was found to bind at 298K with both AAG and HSA with the binding constant of 8.128×103 and 4.120×103 for AAG and HSA, respectively. There is stronger interaction of clofarabine with AAG as compared to HSA. The Gibbs free energy change was found to be negative for the interaction of clofarabine with AAG and HSA indicating that the binding process is spontaneous. Binding of clofarabine with HSA and AAG induced ordered structures in both proteins and lead to molecular compaction. Clofarabine binds to HSA near to drug site II. Hydrogen bonding and hydrophobic interactions were the main bonding forces between HSA-clofarabine and AAG-clofarabine as revealed by docking results. This study suggests the importance of binding of anticancer drug to AAG spatially in the diseases like cancers where the plasma concentration of AAG increases many folds. Design of drug dosage can be adjusted accordingly to achieve optimal treatment outcome.

Preparation and characterization of an aptamer-functionalized solid-phase microextraction fiber and its application in the selective monitoring of adenosine phosphates with liquid chromatography and tandem mass spectrometry.

An aptamer with adenosine triphosphate as a ligand was immobilized onto the surface of a porous-polymer-coated fiber to obtain an aptamer-functionalized porous-polymer-coated solid-phase microextraction fiber. The fiber was observed with a crosslinked and porous morphological surface structure. It shows specific selectivity to adenosine triphosphate with a selectivity coefficient of 22.1 compared to the scrambled oligonucleotide functionalized fiber, and the selectivity factors over other analogues and reference compounds were from 6.1 to 77.5. When the fiber-based solid-phase microextraction was coupled with liquid chromatography and tandem mass spectrometry, detection limits of 2.7, 29, and 34 µg/L were achieved for adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, respectively. The spiking recoveries of 77.6-91.9% were achieved for trace adenosine phosphates in human serum sample. Furthermore, the fibers showed high stability and good reusability and could be used over 50 times for the real serum sample pretreatment without remarkable performance reduction.

A capillary zone electrophoresis method for adenine nucleotides analysis in Saccharomyces cerevisiae.

Adenosine triphosphate and its metabolites are involved in the cellular metabolism process in Saccharomyces cerevisiae. It is very important to simultaneously determine the relative contents of ATP and its metabolites in yeast. In this study, an effective capillary zone electrophoresis method with high selectivity was established. The calibration curves were linear in the concentration range from 1 to 20mg/L (ATP and cAMP) and 2 to 40mg/L (ADP and AMP) with excellent correlation coefficients (r(2))>0.999. The recovery of ATP, ADP, AMP, and cAMP were 99.4%, 94.7%, 100.3% and 99.6%, respectively. Simple sample preparation and easy detection of ATP and its metabolites make this method suitable for the study of changes in the four adenine nucleotides levels caused by caloric restriction in yeast. It is expected that the current method may contribute to further energy metabolism and related investigations of yeast.

Identification of the distribution of adenosine phosphates, nucleosides and nucleobases in royal jelly.

Nucleotides, nucleosides and nucleobases play a greater role in the physiological activity of organisms which are highly present in royal jelly (RJ). The objective of the present study is to develop a HPLC method to simultaneous determine nucleotides, nucleosides and nucleobases in RJ and access them in fresh and commercial RJ samples. The LOD and LOQ were 12.2-99.6 µg/L and 40.8-289.4 µg/L, respectively with nearly 100.9% recoveries. Except uric acid, all other compounds were found in RJ samples. Significant difference in the average content of compounds in fresh (2682.93 mg/kg) and commercial samples (3152.78 mg/kg) were observed. AMP, adenosine and adenine were found predominant in all the samples. Significant higher levels of ATP, ADP and AMP was seen in fresh RJ samples, and IMP, uridine, guanosine, and thymidine was seen in commercial RJ samples. The investigated compounds can be used as indexes for assessment RJ freshness and quality.

Prediction of hepatitis C virus interferon/ribavirin therapy outcome based on viral nucleotide attributes using machine learning algorithms.

BACKGROUND: Hepatitis C virus (HCV) causes chronic hepatitis C in 2-3% of world population and remains one of the health threatening human viruses, worldwide. In the absence of an effective vaccine, therapeutic approach is the only option to combat hepatitis C. Interferon-alpha (IFN-alpha) and ribavirin (RBV) combination alone or in combination with recently introduced new direct-acting antivirals (DAA) is used to treat patients infected with HCV. The present study utilized feature selection methods (Gini Index, Chi Squared and machine learning algorithms) and other bioinformatics tools to identify genetic determinants of therapy outcome within the entire HCV nucleotide sequence. RESULTS: Using combination of several algorithms, the present study performed a comprehensive bioinformatics analysis and identified several nucleotide attributes within the full-length nucleotide sequences of HCV subtypes 1a and 1b that correlated with treatment outcome. Feature selection algorithms identified several nucleotide features (e.g. count of hydrogen and CG). Combination of algorithms utilized the selected nucleotide attributes and predicted HCV subtypes 1a and 1b therapy responders from non-responders with an accuracy of 75.00% and 85.00%, respectively. In addition, therapy responders and relapsers were categorized with an accuracy of 82.50% and 84.17%, respectively. Based on the identified attributes, decision trees were induced to differentiate different therapy response groups. CONCLUSIONS: The present study identified new genetic markers that potentially impact the outcome of hepatitis C treatment. In addition, the results suggest new viral genomic attributes that might influence the outcome of IFN-mediated immune response to HCV infection.

Poly(glycidyl methacrylate-co-N-methylolacrylamide-co-ethylene dimethacrylate) monolith coupled to high-performance liquid chromatography for the determination of adenosine phosphates in royal jelly.

A polymer monolith microextraction method coupled with high-performance liquid chromatography was developed for the determination of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate. The monolithic column was synthesized inside fused-silica capillaries using thermal initiation free-radical polymerization with glycidyl methacrylate as the monomer, ethylene dimethacrylate as the cross-linker, cyclohexanol, and 1-dodecanol as the porogen. N-Methylolacrylamide, an important hydrophilic monomer, was incorporated into the polymerization mixture to enhance the hydrophilicity of the poly(glycidyl methacrylate-co-ethylene dimethacrylate) column. The obtained poly(glycidyl methacrylate-co-N-methylolacrylamide-co-ethylene dimethacrylate) monolith was characterized by scanning electron microscopy, Fourier-transform infrared spectra, and X-ray photoelectron spectroscopy. Optimum conditions for the preconcentration and separation of the target adenosines were also investigated. Under the optimum conditions, we obtained acceptable linearities, low limits of detection, and good relative standard deviations. The developed polymer monolith microextraction with high-performance liquid chromatography method exhibited a good performance with recovery values in the range of 76.9-104.7% when applied to the determination of the adenosines in five royal jelly samples.

Purine metabolite and energy charge analysis of Trypanosoma brucei cells in different growth phases using an optimized ion-pair RP-HPLC/UV for the quantification of adenine and guanine pools.

Human African Trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. Although trypanosomes are well-studied model organisms, only little is known about their adenine and guanine nucleotide pools. Besides being building blocks of RNA and DNA, these nucleotides are also important modulators of diverse biochemical cellular processes. Adenine nucleotides also play an important role in the regulation of metabolic energy. The energetic state of cells is evaluated by the energy charge which gives information about how much energy is available in form of high energy phosphate bonds of adenine nucleotides. A sensitive and reproducible ion-pair RP-HPLC/UV method was developed and optimized, allowing the quantification of guanine and adenine nucleosides/nucleotides in T. brucei. With this method, the purine levels and their respective ratios were investigated in trypanosomes during logarithmic, stationary and senescent growth phases. Results of this study showed that all adenine and guanine purines under investigation were in the low mM range. The energy charge was found to decrease from logarithmic to static and to senescent phase whereas AMP/ATP, ADP/ATP and GDP/GTP ratios increased in the same order. In addition, the AMP/ATP ratio varied as the square of the ADP/ATP ratio, indicating AMP to be the key energy sensor molecule in trypanosomes.

A sensitive LC-MS/MS method for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells.

Clofarabine triphosphate is an intracellular active metabolite of clofarabine. In the present study, we developed and validated a rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood using the Ficoll gradient centrifugation method. Chromatographic separation was performed on a CN column using an isocratic mobile phase comprising acetonitrile/5mM ammonium acetate with 0.001% ammonium hydroxide (20/80, v/v) at a flow rate of 0.60 mL/min. Detection was carried out by MS/MS in the multiple reaction monitoring mode using a negative electrospray ionization interface. The method was validated in concentration ranges of 1.25-100 ng/10(7) cells with acceptable accuracy and precision using 50 µL of cell extract. Clofarabine triphosphate was stable in a series of stability studies with bench-top, auto-sampler, and repeated freeze-thaw cycles. The validated method was successfully used to measure the concentrations of clofarabine triphosphate in PBMCs from cancer patients treated with clofarabine.