Simultaneous detection of creatine and creatinine using a sequential injection analysis/biosensor system

Carbon paste based biosensors for the determination of creatine and creatinine have been integrated into a sequential injection system. Applying the multi-enzyme sequence of creatininase (CA), and/or creatinase (CI) and sarcosine oxidase (SO), hydrogen peroxide has been detected amperometrically. The linear concentration ranges are of pmol/L to nmol/L magnitude, with very low limits of detection. The proposed SIA system can be utilized reliably for the on-line simultaneous detection of creatine and creatinine in pharmaceutical products, as well as in serum samples, with a rate of 34 samples per hour and RSD values better than 0.16% (n=10).     

Rapid determination of gatifloxacin in biological samples and pharmaceutical products using europium‐sensitized fluorescence spectrophotometry

A europium‐sensitized fluorescence spectrophotometry method using an anionic surfactant, sodium dodecyl benzene sulphonate (SDBS), was developed for the determination of gatifloxacin (GFLX). The GFLX–Eu³⁺–SDBS system was studied and it was found that SDBS significantly enhanced the fluorescence intensity of the GFLX–Eu³⁺ complex (about 25‐fold). The optimal experimental conditions were determined as follows: excitation and emission wavelengths of 338 and 617 nm, pH 7.5, 3.0 × 10^–6 mol/L europium(III), and 5.0 × 10^–5 mol/L SDBS. The enhanced fluorescence intensity of the system (ΔI_f) showed a good linear relationship with the concentration of GFLX over the range 1.0 × 10^–8–8.0 × 10^–7 mol/L with a correlation coefficient of 0.9990. The detection limit (S:N = 3) was determined as 1.0 × 10^–9 mol/L. This method has been successfully applied for the determination of GFLX in pharmaceuticals and human urine/serum samples. Compared with most other methods reported, the rapid and simple procedure proposed here offered higher sensitivity, wider linear range and good stability. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

Highly sensitive fluorescence detection of glycoprotein based on energy transfer between CuInS2 QDs and rhodamine B

A highly sensitive fluorescence method for glycoprotein detection has been established based on fluorescence resonance energy transfer (FRET) between CuInS₂ quantum dots (QDs) and rhodamine B (RB). Lectins comprise a group of proteins with unique affinities toward carbohydrate structures, so the process of FRET can occur between lectin‐coated QDs (CuInS₂ QDs–Con A conjugates, acceptors) and carbohydrate‐coated RB (RB–NH₂‐glu conjugates, donors). The fluorescence of lectin‐coated QDs was recovered in the presence of a glycoprotein such as glucose oxidase (GOx) and transferrin (TRF), which significantly reduced the FRET efficiency between the donor and the acceptor. Under optimal conditions, a linear correlation was established between the fluorescence intensity ratio I₆₅₄/I₅₇₇ and the TRF concentration over the range of 6.90 × 10^‐10 to 3.45 × 10^‐8 mol/L, with a detection limit of 2.5 × 10^‐10 mol/L. The linear range for GOx is 3.35 × 10^‐10 to 6.70 × 10^‐8 mol/L, with a detection limit of 1.5 × 10^‐10 mol/L. The proposed method was applied to the determination of glycoprotein in human serum and cell‐extract samples with satisfactory results. Furthermore, CuInS₂ QDs–Con A conjugates are used as safe and efficient optical nanoprobes in HepG2 cell imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Electrophoresis–chemiluminescence detection of phenols catalyzed by hemin

Based on the catalytic activity of hemin, an efficient biocatalyst, an indirect capillary electrophoresis–chemiluminescence (CE‐CL) detection method for phenols using a hemin–luminol–hydrogen peroxide system was developed. Through a series of static injection experiments, hemin was found to perform best in a neutral solution rather than an acidic or alkaline medium. Although halide ions such as Br^? and F^? could further enhance the CL signal catalyzed by hemin, it is difficult to apply these conditions to this CE‐CL detection system because of the self‐polymerization of hemin, as it hinders the CE process. The addition of concentrated ammonium hydroxide to an aqueous/dimethyl sulfoxide solution of hemin–luminol afforded a stable CE‐CL baseline. The indirect CE‐CL detection of five phenols using this method gave the following limits of detections: 4.8 × 10^?8 mol/L (o‐sec‐butylphenol), 4.9 × 10^?8 mol/L (o‐cresol), 5.4 × 10^?8 mol/L (m‐cresol), 5.3 × 10^?8 mol/L (2,4‐dichlorophenol) and 7.1 × 10^?8 mol/L (phenol). Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of arecoline in areca nut based on field amplification in capillary electrophoresis coupled with electrochemiluminescence detection

A sensitive capillary electrophoresis–electrochemiluminescence (CE–ECL) assay with an ionic liquid (IL) was developed for the determination of arecoline in areca nut. The IL, 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMImBF₄), was an effective additive improved not only the separation selectivity but also the detection sensitivity of the analyte. BMImBF₄ in the separation electrolyte made the resistance of the separation buffer much lower than that of the sample solution, which resulted in an enhanced field amplified electrokinetic injection CE. ECL intensity of arecoline is about two times higher than that of the analyte with phosphate–IL buffer system. Resolution between arecoline and other unknown compounds in real samples was improved. Under the optimized conditions (ECL detection at 1.2 V, 16 kV separation voltage, 20 mmol/L phosphate with 10 mmol/L BMImBF₄ buffer at pH 7.50, 5 mmol/L Ru(bpy)₃²⁺ and 50 mmol/L phosphate buffer in the detection reservoir), a detection limit of 5 × 10^–9 mol/L for arecoline was obtained. Relative standard deviations of the ECL intensity and the migration time were 4.51% and 0.72% for arecoline. This method was successfully applied to determination of the amount of arecoline in areca nut within 450 s. Copyright © 2012 John Wiley & Sons, Ltd.     

The determination of glutamine with flow‐injection chemiluminescence detection and mechanism study

The main purpose of this study was to develop an inexpensive, simple, rapid and sensitive chemiluminescence (CL) method for the determination of glutamine (Gln) using a flow‐injection (FI) system. Gln was found to strongly inhibit the CL signal of the luminol–H₂O₂–CuSO₄ system in Na₂B₄O₇ solution. A new FI‐CL method was developed for the determination of Gln. Parameters affecting the reproducibility and CL detection were optimized systematically. Under the optimized conditions, the corresponding linear regression equation was established over the range of 5.0 × 10^?7 to 2.5 × 10^?6 mol/L with the detection limit of 1.8 × 10^?8 mol/L. The relative standard deviation was found to be 1.8% for 11 replicate determinations of 1.5 × 10^?6 mol/L Gln. The proposed method has been satisfactorily applied for the determination of Gln in real samples (Marzulene‐s granules) with recoveries in the range of 98.7–108.6%. The minimum sampling rate was about 100 samples/h. The possible mechanism of this inhibitory CL was studied by fluorescence spectrophotometer and UV–vis spectrophotometer. Copyright © 2009 John Wiley & Sons, Ltd.     

Novel fluorescent sensor using molecularly imprinted silica microsphere‐coated CdSe@CdS quantum dots and its application in the detection of 2,4,6‐trichlorophenol from environmental water samples

In this study, a high fluorescence sensitivity and selectivity, molecularly imprinted nanofluorescent polymer sensor (MIP@SiO₂@QDs) was prepared using a reverse microemulsion method. 2,4,6‐Trichlorophenol (2,4,6‐TCP) was detected using fluorescence quenching. Tetraethyl orthosilicate (TEOS), quantum dots (QDs) and 3‐aminopropyltriethoxysilane (APTS) were used as cross‐linker, signal sources and functional monomer respectively. The sensor (MIP@SiO₂@QDs) and the non‐imprinted polymer sensor (NIP@SiO₂@QDs) were characterized using infra‐red (IR) analysis, X‐ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The selectivity of MIP@SiO₂@QDs was examined by comparing 2,4,6‐TCP with other similar functional substances including 2,4‐dichlorophenol (2,4‐DCP), 2,6‐dichlorophenol (2,6‐DCP) and 4‐chlorophenol (4‐CP). Results showed that MIP@SiO₂@QDs had better selectivity for 2,4,6‐TCP than the other compounds. Fluorescence quenching efficiency displayed a good linear response at the 2,4,6‐TCP concentration range 5–1000 μmol/L. The limit of detection (LOD) was 0.9 μmol/L (3σ, n = 9). This method was equally applicable for testing actual samples with a recovery rate of 98.0–105.8%. The sensor had advantages of simple pretreatment, good sensitivity and selectivity, and wide linear range and could be applied for the rapid detection of 2,4,6‐TCP in actual samples.     

Biosensors for the Determination of Ortho-acetyl-L-carnitine. Their Utilization as Detectors in a Sequential Injection Analysis System

Abstract

In order to determine ortho-acetyl-L-carnitine, two biosensors were proposed. The biosensors were designed using physical immobilization of L-amino acid oxidase (L-AAOD) and horseradish peroxidase (HRP). Electrode characteristics were obtained and compared for the two carbon paste (graphite powder and paraffin oil) biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol/L to nmol/L, magnitude order with low limits of detection. Due to their reliability, the biosensors were used as detectors in a sequential injection analysis system, and gave reliable results for on-line assay of ortho-acetyl-L-carnitine in synthesis process control with a frequency of 75 samples per hour.     

Trace element imbalances in patients undergoing chronic hemodialysis therapy – Report of an observational study in a cohort of Portuguese patients

IntroductionPatients with end-stage renal disease undergoing hemodialysis therapy are at risk of developing deficiencies of essential trace elements and/or overload of toxic trace elements, both of which may significantly affect their clinical status of. Those imbalances may result from the disease itself but also from the quality of the therapeutic process, namely the hemodialysis process, which has greatly evolved in the last decades. Thus, old observations that have been assumed as very well-proven have been recently questioned. In this case-control study we evaluate the current trace elements status in a group of Portuguese patients under hemodialysis therapy.Material and methodsSerum samples from patients (n = 93), collected for the routine periodic control of Al levels, were analyzed for a wide panel of trace elements (Li, Al, Mn, Co, Ni, Cu, Zn, Se, Rb, Sr, Mo, Cd, Ba, Pb) using inductively coupled plasma mass spectrometry technique (hemodialysis patients’ group). For comparison purposes, samples of individuals with no evidence of renal disease according to standard laboratory analytical criteria (n = 50) were also analyzed (control group).ResultsThe results showed significant differences between the two groups, with higher values in hemodialysis patients group for Al (14.6 vs. 9.5 μg/L), Co, Ni, Sr, Mo (4.5 vs. 1.4 μg/L), Cd (0.058 vs. 0.025 μg/L) and Pb (0.55 vs. 0.30 μg/L); and lower values in hemodialysis patients group for Li (4.0 vs. 75.8 μg/L), Mn, Cu (943.5 vs. 1038.5 μg/L), Zn (943.5 vs. 1038.5 μg/L), Se (71.5 vs. 103.8 μg/L), Rb (202.4 vs. 300.3 μg/L) and Ba (0.65 vs. 8.7 μg/L).ConclusionThis study confirms that hemodialysis patients tend to present significant trace elements imbalances, which may be related to the higher morbidity and mortality observed in this specific patients’ group.     

Rapid and Simple Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantifications of Triazole Antifungals in Human Serum

Purpose

To develop and validate a one-step, rapid and simple reversed-phase high-performance liquid chromatography (HPLC)-based protocol for the simultaneous measurement of voriconazole (VCZ), posaconazole (POSA), itraconazole (ITC) in serum/plasma.

Methods

Calibration standards (CS) and quality control samples were prepared in drug-free serum by spiking with the triazoles at different concentrations. HPLC was performed with C₁₈ column, isocratic mobile phase after extraction with cold acetonitrile. The standardized method was tested in 2693 patients’ serum/plasma samples.

Results

Linearity of CS for ITC, VCZ and POSA was proportional to the nominal concentration (correlation coefficient?>?0.999). Limit of detection (mg/L) for ITC, VCZ and POSA was 0.25, 0.25 and 0.125, respectively. The lower limit of quantification (mg/L) for ITC, VCZ and POSA was 0.5, 0.5 and 0.25, respectively. Precision and accuracy were in acceptable range with 100% average percentage recovery. No interferences from endogenous substances and other antimicrobial compounds were noted. In clinical samples, the therapeutic range achieved for VCZ was 39.9%. Whereas, 61.1% and 44% of samples with ITC and POSA, respectively, were in the sub-therapeutic range.

Conclusion

We developed a rapid and simple HPLC method to quantify common triazoles in a single chromatographic run allowing simultaneous measurement of different antifungals in a small volume of serum/plasma. Thus, therapeutic drug monitoring requests can be processed in one run without changing the protocol parameters, column or column conditioning thereby improving turnaround time.

     

Determination of 5‐hydroxytryptamine in serum by electrochemiluminescence detection with the aid of capillary electrophoresis

A rapid, sensitive and simple electrochemiluminescence method for the determination of 5‐hydroxytryptamine (5‐HT) using capillary electrophoresis was proposed. The experimental parameters, including the detection potential, the concentration of Ru(bpy)₃²⁺, the concentration and pH of phosphate buffer for separation and detection, the injection voltage and time and the separation voltage on the determination of 5‐HT, were optimized. Under the optimized conditions, the linear concentration range for 5‐HT was 3.5 × 10^‐9–5.1 × 10^‐3 mol/L, with a detection limit of 5 × 10^‐10 mol/L. The relative standard deviations (RSDs) of the ECL intensity and the migration times for six continuous injections of 1.0 µmol/L 5‐HT were 2.48% and 1.3%, respectively. The method was successfully applied to 5‐HT assay in samples of human serum in 5 min and the extraction recoveries with spiked serum samples were over 94.4%. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of myocardial high-energy phosphates using bioluminescence

The measurement of myocardial high-energy phosphates (HEP) has become essential in the evaluation of current methods of myocardial protection both in the experimental and clinical setting. Assays for high-energy phosphates have required as much as 50 mg of myocardial tissue which prevents repeated biopsies in the clinical setting as well as in the experimental laboratory. Using the reaction of bioluminescence described by McElroy W. D. and B. L. Strehler (1949, Arch. Biol. Chem.22, 420), we have developed a technique to measure both adenosine triphosphate and creatine phosphate on samples of myocardial tissue weighing less than 10 mg. A liquid scintillation counter measures the light produced by ATP when added to a firefly extract containing luciferin/luciferase. The reaction is complete in seconds and is detected in the counter during the first 10-s count. Repeated samples have demonstrated a variation of less than 4% between samples. A 25-μl sample is diluted up to 40 μl of firefly extract for detection of adenosine triphosphate. Creatine phosphate is measured by the in vitro production of adenosine triphosphate which is maximum in 10 min when adenosine diphosphate and creatine kinase are added. Again reproducibility of repeated analyses demonstrates a 4% difference in creatine phosphate values. The rapidity, reproducibility, and ability to use ultramicrosamples allows investigators to analyze high-energy phosphates during various methods of myocardial protection currently used in clinical setting.     

Capillary electrophoresis coupled with end‐column electrochemiluminescence for the determination of ephedrine in human urine,and a study of its interactions with three proteins

A tris(2,2‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)‐based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10^–8–6.0 × 10^–6 g/mL. The detection limit was 4.5 × 10^–9 g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt‐C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt‐C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴ mol/L, 6.58 × 10³ mol/L and 1.59 × 10⁴ mol/L, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Evaluation of lophine derivatives as L‐012 (luminol analog)‐dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2

8‐Amino‐5‐chloro‐7‐phenylpyrido[3,4‐d]pyridazine‐1,4(2H,3H)dione (L‐012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L‐012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine‐based CL enhancers of the horseradish peroxidase (HRP)‐catalyzed CL oxidation of luminol, namely 2‐(4‐hydroxyphenyl)‐4,5‐diphenylimidazole (HDI), 2‐(4‐hydroxyphenyl)‐4,5‐di(2‐pyridyl)imidazole (HPI), 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenylboronic acid (DPA), and 4‐[4,5‐di(2‐pyridyl)‐1H‐imidazol‐2‐yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L‐012 and evaluated these as L‐012‐dependent CL enhancers. In addition, to detect HRP and/or H₂O₂ with higher sensitivity, each detection condition for the L‐012–HRP–H₂O₂ enhanced CL was optimized. All the derivatives enhanced the L‐012‐dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L‐012‐dependent CL using 4‐iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H₂O₂ detection, the detection limits for enhanced CL with HPI and 4‐iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L‐012‐dependent CL enhancer. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.     

Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L‐T3, L‐T4, and D‐T4

A fast screening method of whole blood was proposed for enantiorecognition of free L‐T₃ , L‐T₄, and D‐T₄. Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L‐T₃ , L‐T₄, and D‐T₄. For the enantiorecognition of free L‐T₄ and D‐T₄ in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L‐T₃ (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L‐T4, and D‐T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP‐based microsensor. For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). Free L‐T₃ , L‐T₄, and D‐T₄ were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%). Chirality 27:973–978, 2015. © 2015 Wiley Periodicals, Inc.     

Serum creatine is not a reliable marker of muscular fitness in young adults

Purpose: Elevated serum creatine and higher handgrip strength are individually associated with better health profiles yet the link between two variables remains unknown. In this cross-sectional study, we evaluated serum creatine levels in relation to handgrip strength in a cohort of 130 young healthy adults (61 women and 69 men; age 23.3?±?2.6?years), while controlling for age, gender, fat-free mass and biomarkers of creatine metabolism as effect modifiers.

Materials and methods: Serum creatine, creatinine and guanidinoacetic acid (GAA) levels were measured with liquid chromatography-tandem mass spectroscopy, while handgrip strength was assessed with a hydraulic hand dynamometer.

Results: Hierarchical multiple regression revealed that our model as a whole explained 79.9% of the variance in handgrip strength (p?p?p?>?0.05).

Conclusions: Having higher blood creatine appears to be unrelated with better physical performance in young healthy adults. Serum creatine was not a reliable marker of muscular fitness in this population.     

A combination of ultrasonic-assisted extraction with RRLC-QQQ method for the determination of artemisinin in the Chinese herb Artemisia annua L

Introduction – Artemisinin, the primary active ingredient of the Chinese herb Artemisia annua L., is known to have considerable anti‐malaria properties. However, rapid, sensitive and selective method for the determination of artemisinin in it is not currently available. Objective – To develop and validate an efficient method for extraction and analysis of artemisinin from the plant samples of Artemisia annua L. by rapid resolution liquid chromatography triple quadrupole mass spectrometry (RRLC‐QQQ). Methodology – Following ultrasound‐assisted extraction (USE), RRLC‐QQQ was utilised to separate and determine artemisinin from the plant sample of Artemisia annua L. The LC separation, QQQ‐MS detection and multiple reaction monitoring (MRM) mode were optimised, and the method validation concluding selectivity, calibration, accuracy and precision, and recovery were also evaluated. Results – LC separation was performed with an isocratic elution of 20% of methanol–water (10 mmol/L ammonium acetate, pH 4.0) on a C₁₈ column. The triple quadrupole MS detection was carried out under MRM mode of precursor ion [M + H]⁺ → fragment ions m/z 265.1 and m/z 247.2. The limits of detection and quantitation of artemisinin were 0.20 and 0.75 ng/mL, respectively. The intra‐ and inter‐day precisions did not exceed 3.71%, and the deviation of the intra‐ and inter‐day mean values did not exceed ±7.50. The average recoveries for artemisinin ranged from 92.45 to 103.8% with an RSD from 2.47 to 2.79%. Conclusion – The developed RRLC‐QQQ assay is an efficient method for separation and determination of artemisinin from the plant samples of Artemisia annua L. Copyright © 2011 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence determination of diazepam by oxidation with N‐bromosuccinimide

A rapid and sensitive flow‐injection chemiluminescence (FI–CL) method is described for the determination of diazepam based on its reaction with N‐bromosuccinimide (NBS) in alkaline medium in the presence of dichlorofluorescein (DCF) as an effective energy‐transfer agent. Under optimum conditions, the proposed method allowed the measurement of diazepam over the range of 2.0 × 10^?6 to 2.0 × 10^?4 mol/L with a detection limit of 5.0 × 10^?7 mol/L. The relative standard deviation for 11 parallel measurements of 2.0 × 10^?5 mol/L diazepam was 2.1%. The method was applied satisfactorily for the determination of diazepam in pharmaceutical preparations, and the results agree well with those obtained by spectrophotometry. The use of the proposed system for the determination of diazepam in urine and plasma samples was also tested. The possible mechanism of the chemiluminescence reaction is discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.     

Lyn regulates creatine uptake in an imatinib-resistant CML cell line

BackgroundImatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells.MethodsStable isotope metabolic labeling, media creatine depletion, and Na⁺/K⁺-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na⁺/K⁺-ATPase and creatine uptake.ResultsInhibition of the Na⁺/K⁺-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na⁺/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na⁺/K⁺-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na⁺/K⁺-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na⁺/K⁺-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells.ConclusionsThese data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na⁺/K⁺-ATPase pump activity.General significanceThese studies identify kinase regulation of the Na⁺/K⁺-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.     

Sensitive determination of 2‐methoxyestradiol in pharmaceutical preparations and serum samples using flow injection chemiluminescence

A rapid and sensitive flow injection chemiluminescence (FI–CL) method is described for the determination of 2‐methoxyestradiol (2ME) based on enhancement of the CL intensity from a potassium ferricyanide–calcein system in sodium hydroxide medium. The optimum conditions for the CL emission were investigated. Under optimized conditions, a linear calibration graph was obtained over the range 1.0 × 10^‐8 to 1.0 × 10^‐6 mol/L (r = 0.998) 2ME with a detection limit (3σ) of 5.4 × 10^‐9 mol/L. The relative standard deviation (RSD) for 5.0 × 10^‐7 mol/L 2ME was 1.7%. As a preliminary application, the proposed method was successfully applied to the determination of 2ME in injection solutions and serum samples. The possible CL mechanism was also proposed. Copyright © 2013 John Wiley & Sons, Ltd.