Human Blood Levels Research Articles

Ultra performance liquid chromatography tandem mass spectrometry assay for determination of kukoamine B in human blood and urine.

In this paper, we report a sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method which is capable of quantifying kukoamine B (KB) levels in human blood and urine. Following solid phase extraction and direct dilution process, the analyte and its internal standard (D5-KB) run on an Acquity UPLC(®) HSS T3 column (2.1×50mm i.d., 1.8μm) by using a gradient elution method (run time was 1.5min). The mass spectrometric analysis was performed by using an API-5500 mass spectrometer coupled with an electro-spray ionization source. The MRM transitions of m/z 531.3(+)→222.1(+) and 536.3(+)→222.1(+) were used to quantify KB and D5-KB respectively. This assay method has been fully validated in terms of selectivity, linearity, lower limit of quantification, precision, accuracy, stability, recovery and matrix effect. The concentration range of this method is 10.0-2000.0ngmL(-1) in blood and 0.5-500.0ngmL(-1) in urine. Linearity (R(2)) of calibration curves were 0.9964±0.0022 and 0.9935±0.0053 for blood and urine, respectively (regression equation: y=ax+b). The precision (RSD%) of quality control samples is less than 10.3% for blood and less than 10.5% for urine. The accuracy (RE%) is within -4.0-11.3% and -11.7-12.5% for blood and urine respectively. KB was stable after 4h in ice-water bath, 1 freeze/thaw cycles and 180days at -80°C for blood samples; and was stable after 3h at room temperature, 3 freeze/thaw cycles and 180days at -80°C for urine samples. Recoveries of KB were 4.7±0.9% in blood and 96.5±1.3% in urine, respectively. Additionally, the applicability of this method has been proved by analyzing clinical samples from pharmacokinetic study of KB in human.

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide's concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p <0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p = 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p = 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide's concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for (125)I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p<0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

Optimization of a Liquid Crystal‐based Sensory Platform for Monitoring Enzymatic Glucose Oxidation

Managing glucose levels in human blood is extremely important for the treatment of diabetes. Here, an innovative sensory strategy has been developed to monitor the enzymatic activities of glucose and glucose oxidase by using confined liquid crystal (LC) birefringent droplet patterns. Acidic products released during the glucose oxidation process lead to a slight decrease in the pH of aqueous systems that can be monitored by pH‐sensitive LC materials. Of the existing pH‐sensitive LC materials, dodecanoic acid‐doped 4‐cyano‐4′‐pentylbiphenyl is inexpensive and easily adjusted to satisfy the 7.4 ± 0.05 pH requirement of human blood. Moreover, the orientational alignment of capillary‐confined pH‐responsive LCs can be disrupted at the aqueous/LC interface following a slight decrease in the critical pH of aqueous reaction systems, which results in an optical signal that can be observed with the naked eye by using polarizing optical microscopy. Based on the stable LC droplet patterns generated by the cylindrical confinement system, the functionalized LCs can selectively detect glucose at concentrations as low as 0.1 pM. This study further advances the previously reported LC‐based glucose monitoring systems by reducing production costs and instituting a smarter LC sensory design. This improved system shows potential for the use in clinical bioassay applications.

Measurement of Circulating Filarial Antigen Levels in Human Blood with a Point-of-Care Test Strip and a Portable Spectrodensitometer.

The Alere Filariasis Test Strip (FTS) is a qualitative, point-of-care diagnostic tool that detects Wuchereria bancrofti circulating filarial antigen (CFA) in human blood, serum, or plasma. The Global Program to Eliminate Lymphatic Filariasis employs the FTS for mapping filariasis-endemic areas and assessing the success of elimination efforts. The objective of this study was to explore the relationship between the intensity of positive test lines obtained by FTS with CFA levels as determined by enzyme-linked immunosorbent assay (ELISA) with blood and plasma samples from 188 individuals who live in a filariasis-endemic area. The intensity of the FTS test line was assessed visually to provide a semiquantitative score (visual Filariasis Test Strip [vFTS]), and line intensity was measured with a portable spectrodensitometer (quantitative Filariasis Test Strip [qFTS]). These results were compared with antigen levels measured by ELISA in plasma from the same subjects. qFTS measurements were highly correlated with vFTS scores (ρ = 0.94; P < 0.001) and with plasma CFA levels (ρ = 0.91; P < 0.001). Thus, qFTS assessment is a convenient method for quantifying W. bancrofti CFA levels in human blood, which are correlated with adult worm burdens. This tool may be useful for assessing the impact of treatment on adult filarial worms in individuals and communities.

Treatment of blood with a pathogen reduction technology using ultraviolet light and riboflavin inactivates Ebola virus in vitro.

Transfusion of plasma from recovered patients after Ebolavirus (EBOV) infection, typically called "convalescent plasma," is an effective treatment for active disease available in endemic areas, but carries the risk of introducing other pathogens, including other strains of EBOV. A pathogen reduction technology using ultraviolet light and riboflavin (UV+RB) is effective against multiple enveloped, negative-sense, single-stranded RNA viruses that are similar in structure to EBOV. We hypothesized that UV+RB is effective against EBOV in blood products without activating complement or reducing protective immunoglobulin titers that are important for the treatment of Ebola virus disease (EVD). Four in vitro experiments were conducted to evaluate effects of UV+RB on green fluorescent protein EBOV (EBOV-GFP), wild-type EBOV in serum, and whole blood, respectively, and on immunoglobulins and complement in plasma. Initial titers for Experiments 1 to 3 were 4.21 log GFP units/mL, 4.96 log infectious units/mL, and 4.23 log plaque-forming units/mL. Conditions tested in the first three experiments included the following: 1-EBOV-GFP plus UV+RB; 2-EBOV-GFP plus RB only; 3-EBOV-GFP plus UV only; 4-EBOV-GFP without RB or UV; 5-virus-free control plus UV only; and 6-virus-free control without RB or UV. UV+RB reduced EBOV titers to nondetectable levels in both nonhuman primate serum (≥2.8- to 3.2-log reduction) and human whole blood (≥3.0-log reduction) without decreasing protective antibody titers in human plasma. Our in vitro results demonstrate that the UV+RB treatment efficiently reduces EBOV titers to below limits of detection in both serum and whole blood. In vivo testing to determine whether UV+RB can improve convalescent blood product safety is indicated.

Inter-Individual Differences in RNA Levels in Human Peripheral Blood.

Relatively little is known about the range of RNA levels in human blood. This report provides assessment of peripheral blood RNA level and its inter-individual differences in a group of 35 healthy humans consisting of 25 females and 10 males ranging in age from 50 to 89 years. In this group, the average total RNA level was 14.59 μg/ml of blood, with no statistically significant difference between females and males. The individual RNA level ranged from 6.7 to 22.7 μg/ml of blood. In healthy subjects, the repeated sampling of an individual’s blood showed that RNA level, whether high or low, was stable. The inter-individual differences in RNA level in blood can be attributed to both, differences in cell number and the amount of RNA per cell. The 3.4-fold range of inter-individual differences in total RNA levels, documented herein, should be taken into account when evaluating the results of quantitative RT-PCR and/or RNA sequencing studies of human blood. Based on the presented results, a comprehensive assessment of gene expression in blood should involve determination of both the amount of mRNA per unit of total RNA (U / ng RNA) and the amount of mRNA per unit of blood (U / ml blood) to assure a thorough interpretation of physiological or pathological relevance of study results.

Targeting G-Quadruplex DNA Structures by EMICORON Has a Strong Antitumor Efficacy against Advanced Models of Human Colon Cancer.

We previously identified EMICORON as a novel G-quadruplex (G4) ligand showing high selectivity for G4 structures over the duplex DNA, causing telomere damage and inhibition of cell proliferation in transformed and tumor cells. Here, we evaluated the antitumoral effect of EMICORON on advanced models of human colon cancer that could adequately predict human clinical outcomes. Our results showed that EMICORON was well tolerated in mice, as no adverse effects were reported, and a low ratio of sensitivity across human and mouse bone marrow cells was observed, indicating a good potential for reaching similar blood levels in humans. Moreover, EMICORON showed a marked therapeutic efficacy, as it inhibited the growth of patient-derived xenografts (PDX) and orthotopic colon cancer and strongly reduced the dissemination of tumor cells to lymph nodes, intestine, stomach, and liver. Finally, activation of DNA damage and impairment of proliferation and angiogenesis are proved to be key determinants of EMICORON antitumoral activity. Altogether, our results, performed on advanced experimental models of human colon cancer that bridge the translational gap between preclinical and clinical studies, demonstrated that EMICORON had an unprecedented antitumor activity warranting further studies of EMICORON-based combination treatments.

A novel multi-parameter assay to dissect the pharmacological effects of different modes of integrin αLβ2 inhibition in whole blood.

The integrin αLβ2 plays central roles in leukocyte adhesion and T cell activation, rendering αLβ2 an attractive therapeutic target. Compounds with different modes of αLβ2 inhibition are in development, currently. Consequently, there is a foreseeable need for bedside assays, which allow assessment of the different effects of diverse types of αLβ2 inhibitors in the peripheral blood of treated patients. Here, we describe a flow cytometry-based technology that simultaneously quantitates αLβ2 conformational change upon inhibitor binding, αLβ2 expression and T cell activation at the single-cell level in human blood. Two classes of allosteric low MW inhibitors, designated α I and α/β I allosteric αLβ2 inhibitors, were investigated. The first application revealed intriguing inhibitor class-specific profiles. Half-maximal inhibition of T cell activation was associated with 80% epitope loss induced by α I allosteric inhibitors and with 40% epitope gain induced by α/β I allosteric inhibitors. This differential establishes that inhibitor-induced αLβ2 epitope changes do not directly predict the effect on T cell activation. Moreover, we show here for the first time that α/β I allosteric inhibitors, in contrast to α I allosteric inhibitors, provoked partial downmodulation of αLβ2, revealing a novel property of this inhibitor class. The multi-parameter whole blood αLβ2 assay described here may enable therapeutic monitoring of αLβ2 inhibitors in patients' blood. The assay dissects differential effect profiles of different classes of αLβ2 inhibitors.

Organophosphorus Inhibition and Characterization of Recombinant Guinea Pig Acetylcholinesterase.

Organophosphorus (OP) pesticides and nerve agents have been designed to inhibit the hydrolysis of the neurotransmitter acetylcholine by covalently binding to the active site serine of acetylcholinesterase while Alzheimer drugs and prophylactics, such as tacrine, are characterized by reversible binding. Historically, the guinea pig has been believed to be the best non-primate model for OP toxicology and medical countermeasure development because, similarly to humans, guinea pigs have low amounts of circulating OP metabolizing carboxylesterase. To explore the hypothesis that guinea pigs are the appropriate responder species for OP toxicology and medical countermeasure development, guinea pig acetylcholinesterase (gpAChE) was cloned into pENTR/D-TOPO, recombined into pT-Rex-DEST30 and expressed in Human Embryonic Kidney 293 cells. Recombinant gpAChE was purified to a specific activity of 800 U/mg using size exclusion and immobilized nickel affinity chromatography, with purity confirmed by gel electrophoresis. Ellman's assay was used to enzymatically characterize gpAChE, identifying a K(M) of 154±18.7 µmol L(-1) and a k(cat) of 4.79x10(4)±5.26x10(2) /sec. Apparent gpAChE IC50's for diisopropylfluorophosphate, dicrotophos, paraoxon, and an Alzheimer's drug, tacrine, were found to be 10.1±1.98, 337±108, 1.02±0.29 and 0.30±0.01 µmol L(-1), respectively. Apparent gpAChE inhibition constants for diisopropylfluorophosphate, dicrotophos, paraoxon, and tacrine were found to be 8.40±0.60, 4.50±0.30, 0.29±0.01 and 0.42±0.07 µmol L(-1), respectively. Lineweaver-Burk plots confirmed tacrine as a mixed inhibitor and paraoxon, dicrotophos and diisopropylfluorophosphate as irreversible non-competitive inhibitors. gpAChE bimolecular rate constants for diisopropylfluorophosphate, dicrotophos and paraoxon were found to be 1.44±0.33x10(4), 1.56±0.12x10(3) and 4.57± 0.23x10(5) L µmol(-1) min(-1), respectively. Although the blood levels of OP metabolizing carboxylesterases in the guinea pig are similar to the low levels in human blood, the gpAChE is different in its enzymology. Therefore, medical countermeasures against OP intoxication should be tested for efficacy with the recombinant form of gpAChE prior to initiating animal studies.

Synergistic effect of bimetallic Ag@Cu nanorods modified electrode for enhanced electrochemical sensing of thiocyanate ions

The thiocyanate (SCN−) level in human saliva, serum, and blood has been considered as a biomarker for analyzing the practice of cigarette smokers. Here, we have developed a highly sensitive and selective electrochemical sensor for SCN− in phosphate buffer using an Ag@Cu bimetallic nanorods (NRDs) modified glassy carbon electrode (GCE). The bimetallic Ag@Cu NRDs were grown on the GCE surface by a simple seed-mediated growth method. Initially, Ag seeds were deposited on the Nafion coated GCE. The deposited Agseeds in turn accelerate the growth of bimetallic Ag@Cu NRDs in the presence of cetyltrimethyl ammonium bromide, which acts as a soft template for the one-dimensional growth of NRDs. The synthesised NRDs were characterized by scanning electron microscopy and energy dispersive X-ray spectroscopy measurements. The Ag@Cu NRDs modified electrode shows an excellent response towards the electrochemical oxidation of SCN−. Cyclic voltammetry was used to study the linear correlation of SCN− oxidation between 1 and 10 mM, and, subsequently, 10 nM was achieved as the detection limit with a signal to noise ratio (S/N = 3) for Ag@Cu bimetallic NRDs modified GCE. This unique sensor shows a high selectivity towards trace amounts of SCN− in the presence of various interfering cations and anions. It satisfactorily recognized the generated SCN− in a real-time application such as in human saliva. Finally, the fingerprint of SCN− existence in the saliva samples of cigarette smokers and non-smokers were differentiated in a short span of time with ease.

Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13.

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.

MnO2-Nanosheet-Modified Upconversion Nanosystem for Sensitive Turn-On Fluorescence Detection of H2O2 and Glucose in Blood.

Blood glucose monitoring has attracted extensive attention because diabetes mellitus is a worldwide public health problem. Here, we reported an upconversion fluorescence detection method based on manganese dioxide (MnO2)-nanosheet-modified upconversion nanoparticles (UCNPs) for rapid, sensitive detection of glucose levels in human serum and whole blood. In this strategy, MnO2 nanosheets on the UCNP surface serve as a quencher. UCNP fluorescence can make a recovery by the addition of H2O2, which can reduce MnO2 to Mn(2+), and the glucose can thus be monitored based on the enzymatic conversion of glucose by glucose oxidase to generate H2O2. Because of the nonautofluorescent assays offered by UCNPs, the developed method has been applied to monitor glucose levels in human serum and whole blood samples with satisfactory results. The proposed approach holds great potential for diabetes mellitus research and clinical diagnosis. Meanwhile, this nanosystem is also generalizable and can be easily expanded to the detection of various H2O2-involved analytes.

A beating heart cell model to predict cardiotoxicity: Effects of the dietary supplement ingredients higenamine, phenylethylamine, ephedrine and caffeine

Some dietary supplements may contain cardiac stimulants and potential cardiotoxins. In vitro studies may identify ingredients of concern. A beating human cardiomyocyte cell line was used to evaluate cellular effects following phenylethylamine (PEA), higenamine, ephedrine or caffeine treatment. PEA and higenamine exposure levels simulated published blood levels in humans or animals after intravenous administration. Ephedrine and caffeine levels approximated published blood levels following human oral intake. At low or midrange levels, each chemical was examined plus or minus 50 µM caffeine, simulating human blood levels reported after consumption of caffeine-enriched dietary supplements. To measure beats per minute (BPM), peak width, etc., rhythmic rise and fall in intracellular calcium levels following 30 min of treatment was examined. Higenamine 31.3 ng/ml or 313 ng/ml significantly increased BPM in an escalating manner. PEA increased BPM at 0.8 and 8 µg/ml, while 80 µg/ml PEA reduced BPM and widened peaks. Ephedrine produced a significant BPM dose response from 0.5 to 5.0 µM. Caffeine increased BPM only at a toxic level of 250 µM. Adding caffeine to PEA or higenamine but not ephedrine further increased BPM. These in vitro results suggest that additional testing may be warranted in vivo to further evaluate these effects.

Organochlorine levels in human blood from residents in areas of limited pesticide use in Sudan

Ninety-nine human blood samples were collected from the riverine region of northern Sudan and the traditional and mechanized rain-fed areas of western and eastern Sudan, representing areas of limited pesticide use in Sudan. Blood samples were analyzed for organochlorine pesticides by gas liquid chromatography (GLC) followed by electron-capture detection (ECD). p,p′-DDE (a metabolite of DDT), heptachlor epoxide, β-hexachlorocyclohexane (β-HCH), and dieldrin were detected in all locations surveyed. The level of total organochlorine burden was highest in the traditional rain-fed area, followed by the mechanized rain-fed area, and the riverine area. A highly significant correlation was observed between total organochlorine blood burden and the age of the donors (r = 0.608**).

Origin of Low Detection Limit and High Selectivity of Roche Accu‐Chek Test Strips that Enables Measurement of Tear Glucose Levels

AbstractTear glucose measurements have been suggested as a potential alternative to blood glucose monitoring for diabetic patients. While previous work has reported a correlation between blood and tear glucose levels in humans, this link has not been thoroughly established and additional clinical studies are needed. We recently reported that Roche’s electrochemical Accu‐Chek blood glucose test strips exhibit far superior analytical performance over other commercial brands, with the low detection limit and high selectivity required for quantitating tear glucose levels. Herein, we evaluate the origin of the high sensitivity and selectivity of the Roche test strips and show that the use of pyrroloquinoline quinone (PQQ)‐dependent glucose dehydrogenase (GDH) as an active enzyme reagent in combination with a nitrosoaniline derivative as an electron transfer mediator are primarily responsible for the low limit of quantification (LOQ) (ca. 9 µM) and enhanced selectivity achieved with these strips.

Effects of triglycerides levels in human whole blood on the extraction of 19 commonly used drugs using liquid–liquid extraction and gas chromatography–mass spectrometry

Liquid–liquid extraction (LLE) is the most commonly sample preparation procedure used by forensic toxicologists in China for screening drugs in whole human blood. It extracts numerous substances from blood including targeted drugs and interfering substances, specifically triglycerides (TG). With increasing prevalence of hyperlipidemia, the influences of TG on LLE and on subsequent analysis with gas chromatography–mass spectrometry (GC–MS) may become a major issue for forensic laboratories. This study aims to elucidate the influences of TG on LLE and to provide possible solutions to this problem. Nineteen commonly encountered drugs in forensic cases were spiked to human whole blood with different TG concentrations. Diethyl ether, ethyl acetate/hexane mixed solutions, chlorobutane and several other frequently used solvents were tested for the extraction of drugs from spiked whole blood. The supernatant organic layer was evaporated to dryness and reconstituted with methanol. The resultant products were analyzed by GC–MS, and the extraction recovery was calculated. LLE with diethyl ether, ethyl acetate/hexane (9:1) and chlorobutane all possessed effective and reliable extraction recoveries for blood sample with low TG concentrations (0.63–6.85mmol/L). At high TG concentrations, diethyl ether produced a highly turbid substance that could not be further analyzed using GC–MS. Extraction recoveries drastically dropped for ethyl acetate/hexane (9:1) mixture at high TG concentrations, while chlorobutane experienced minimal drops in extraction recoveries. In conclusion, TG levels in whole blood noticeably influence drug recovery to variable extents depending on the LLE solvent. Chlorobutane showed minimal influences from TG content in whole blood and thus is the recommended LLE solvent for forensic drug extraction.

Exercise increases sphingoid base-1-phosphate levels in human blood and skeletal muscle in a time- and intensity-dependent manner.

PurposeSphingosine-1-phosphate (S1P) regulates cardiovascular function and plays an important role in muscle biology. We have previously reported that cycling exercise increased plasma S1P. Here, we investigated the effect of exercise duration and intensity on plasma and skeletal muscle S1P levels.MethodsIn the first experiment, 13 male athletes performed a 60-min exercise at 65 % of VO2max and a graded exercise until exhaustion on a rowing ergometer. Samples of the venous blood were taken, and plasma, erythrocytes and platelets were isolated. In the second experiment, ten male moderately active subjects performed three consecutive periods of one-leg knee extension exercise (at 25, 55 and 85 % of the maximal workload). Muscle biopsies and blood samples from the radial artery and femoral veins were taken.ResultsUnder basal conditions, S1P was released from the leg, as its concentration was lower in the arterial than in the venous plasma (p < 0.01). Exercise until exhaustion increased plasma S1P and sphinganine-1-phosphate (SA1P) concentration (p < 0.05), whereas moderate-intensity exercise elevated only SA1P (p < 0.001). Although knee extension increased muscle S1P content (p < 0.05), it was not released but taken up across the leg during exercise. However, sphingosine was released from both working and resting leg at the highest workload (p < 0.05).ConclusionsPlasma S1P concentration is elevated only by high-intensity exercise which results, at least in part, from increased availability of sphingosine released by skeletal muscle. In addition, exercise markedly affects S1P dynamics across the leg. We speculate that S1P may play an important role in adaptation of skeletal muscle to exercise.Electronic supplementary materialThe online version of this article (doi:10.1007/s00421-014-3080-x) contains supplementary material, which is available to authorized users.

Development of Non-enzymatic Electrochemical Glucose Sensor Based on Graphene Oxide Nanoribbon – Gold Nanoparticle Hybrid

This study focuses on enhancing the catalytic activity of gold nanoparticles (AuNPs) by using graphene oxide nanoribbons (GONRs) as a functional supporting matrix for a non-enzymatic glucose sensor operating under neutral condition. The proposed method for electrode fabrication offers the ability to control the GONR and AuNP loading for optimum catalytic performance. In our catalytic system, AuNPs supported by GONRs was profoundly superior to the unsupported conventional bare gold electrode, with a greatly enhanced current density (≈200%). This is attributed not only to the high total surface area of the AuNPs compared to that of a Au sheet, but also to the three-dimensional specific interaction between the functional groups on the GONRs and the Au active sites with the reactant and the intermediates that promote the reaction kinetics. The sensitivity of the AuNP/GONR hybrid electrode was examined through chronoamperometry and linear sweep voltammetry methods, and achieved a linear range from 0.5μM to 10mM, covering the normal glucose level in human blood. The poisoning effect of chloride ions was overcome by utilising an optimised polypyrrole-Nafion membrane, opening the possibility of the application of our electrode in continuous glucose monitoring for diabetic patients.

Novel Cholesterol Sensor Based on Ultra-Low Detection Limit Hydrogen Peroxide Sensor

Hydrogen peroxide attracts a great interest due to its important role in food, pharmaceutical, and clinical applications. Hydrogen peroxide is also a by-product in many enzyme catalytic reactions, such as glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, urate oxidase, aldehyde oxidase, and oxalate oxidase, which are implemented to detect glucose, lactic acid, cholesterol, ethanol, urea, formaldehyde, and oxalate, respectively. These biomolecules are significant markers in many biologically metabolic reactions. In our study, we present a cholesterol sensor based on a high sensitivity hydrogen peroxide sensor with ultra-low detection limit. The sensor can directly test the sample and only need very small amount of sample. And because of the simple structural design and fabrication, the sensor can be used as a cheap, efficient, and portable sensor system. The HRP-modified resistive sensors based on n-alkylated polyaniline(PANI) detect hydrogen peroxide in solution with very high sensitivity, ultra-low limit, and short response time. The sensitivity is higher than that of other sensing methods, such as electrochemical sensors or transistor sensors. The detection limit of PANI sensor is 0.7 nM. It is three orders smaller than that of other common methods with detection limit around 1 μM. To the best of our knowledge, it is the lowest detection limit that has ever been reported. And we combine the hydrogen peroxide sensor with cholesterol oxidase to build up the cholesterol sensor. In our study, the PANI layer and gold electrodes were deposited on silicon nitride substrate, and the PANI was applied to fabricate a thin film between two electrodes. Then the PANI layer was sultonated by propane sultone and modified with HRP. After that, the device was combined with dialysis membrane which was modified with cholesterol oxidase. During the measurement, the sensor was operated at 100mV and different concentrations of cholesterol PBS solutions (pH=7.0) were dropped on it. The cholesterol reacted with the cholesterol oxidase and created hydrogen peroxide on the PANI thin film (Figure 1). The current change was measured when the hydrogen peroxide reacted with the HRP immobilized PANI thin film. According to regular cholesterol level in human blood, we tested the cholesterol solution from 100 mg/dl to 400 mg/dl ,and we get a very good linear result (Figure 2). In summary, the cholesterol sensor can provide a more exact cholesterol concentration detection. The simple process for the sensor fabrication also allows the sensor to be cheap, disposable and combinable with other sensors to build up sensing system. This work was partially supported by National Science Council grant (No.99B20495A & 101-2221-E-007-102-MY3) and by the research grant (100N2049E1) at National Tsing Hua University.