Polymeric thiols as enzyme activators of serum creatine phosphokinase

Several hydrophilic polymeric thiols were prepared from aminoactivated polymeric supports by reaction with N-acetylhomocysteinethiolactone. Supports include agaroses, cellulose, Glycophase™ controlled-pore glass, and Matrex™ acrylic beads. Thiol content in these polymers was 3–72 μmol SH/g dry polymer. Several were effective solid-phase activators of the sulfhydryl-dependent enzyme creatine phosphokinase at concentrations comparable to that of monomeric thiol required for enzyme activation. The kinetic activation curves for the polymeric and the monomeric (thioglucose) activators were similar, suggesting unhindered interaction of the enzyme with the polymeric activator.     

GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine

In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-methyl-5H-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measurement of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and trifluoroacetylation of creatinine and creatine are the oldest reported derivatization methods for their GC-mass spectrometry (MS) analysis in human serum using flame- or electron-ionization. We performed GC-MS studies on the derivatization of creatinine (d₀-creatinine), [methylo-²H₃]creatinine (d₃-creatinine, internal standard) and creatine (d₀-creatine) with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) using standard derivatization conditions (60 min, 60 °C), yet in the absence of any base. Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form several derivatives. Their early eluting N,N,O-tris(trimethylsilyl) derivatives (8.9 min) were found to be useful for the precise and accurate measurement of the sum of creatinine and creatine in human urine (10 µL, up to 20 mM) by selected-ion monitoring (SIM) of m/z 271 (d₀-creatinine/d₀-creatine) and m/z 274 (d₃-creatinine) in the NICI mode. In the PICI mode, SIM of m/z 256, m/z 259, m/z 272 and m/z 275 was performed. BSTFA derivatization of d₀-creatine from a freshly prepared solution in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min), presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min) and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine. Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone.     

Molecularly imprinted solid-phase extraction combined with molecularly imprinted polymer-sensor: a diagnostic tool applicable to creatine deficiency syndrome

Primary creatine deficiency syndromes (CDS) are a new group of disorders caused by guanidinoacetate methyltransferase (GAMT) deficiency, which affects endogenous creatine biosynthesis with depletion of body creatine. A deficiency in creatine can be corrected by treatment with oral creatine supplementation and this necessitates a simple and sensitive screening method for early detection of creatine in dilute physiologic fluids. In this work an artificial receptor, molecularly imprinted polymer (MIP), for creatine was used both as a material for solid-phase extraction (SPE) and as a sensing element in a voltammetric sensor. Using the combination of molecularly imprinted solid-phase extraction (MISPE) with a complementary MIP sensor, the minimum detectable amount was found to be 0.0015 ng mL(-1) (RSD = 1.3%, S/N = 3). The MISPE-MIP sensor combination provided up to 60-fold preconcentration, which was more than sufficient for achieving the required quantification limit 50 ng mL(-1) (or 0.0025 ng mL(-1) after 2 x 10(4)-fold dilution) for creatine in human blood serum.     

Preparation and Properties of a Micro Enzyme Sensor for Creatine

Abstract

A micro enzyme sensor using creatine amidinohydrolase (EC 3. 5. 3. 3) and sarcosine oxidase (EC 1. 5. 3. 1) was developed for measuring creatine. The sensor consisted of platinum and silver wires fixed in a glass tubing with epoxy resin adhesive, and creatine amidinohydrolase and sarcosine oxidase immobilized on their tip by cross-linking method. The calibration curve for measurement of creatine, the effects of pH, temperature, the amount of flavin adenine dinucleotide and compounds on the sensitivity, and the life-time of the sensor were examined. Under optimal conditions, the response of the sensor was linear in the concontration range of 10 μ M ～ 0.65 mM creatine.     

Effect of exercise on the creatine resonances in 1H MR spectra of human skeletal muscle

1H MR spectra of human muscles were recorded before, during, and after fatiguing exercise. In contrast to expectations, it was found that the spectral contributions of creatine/phosphocreatine (Cr/PCr) were subject to change as a function of exercise. In particular, the dipolar-coupled methylene protons of Cr/PCr were found to be reduced in intensity in proportion to the co-registered PCr levels. Recovery after exercise and behavior under ischemic conditions provide further evidence to suggest that the contributions of the CH2 protons of Cr/PCr to 1H MR spectra of human muscle in vivo reflect PCr rather than Cr levels. Variation of experimental parameters showed that this effect is not due to a trivial change in relaxation times. At present it can only be speculated about why the Cr resonances have reduced NMR visibility. If temporary binding to macromolecules should be involved, the free Cr concentration-important for equilibrium calculations of the creatine kinase reaction-might be different from what was previously assumed.     

Determination of telbivudine in the plasma of chronic hepatitis B patients in long‐term treatment by high‐performance liquid chromatographic–tandem mass spectrometry

Creatine kinase elevation is commonly reported in telbivudine‐treated patients. However, little is known about the relationship between this adverse drug reaction and plasma concentration. In this study, a sensitive, rapid and safe quantitative bioanalytical method has been established by using LC‐MS/MS for the determination of telbivudine in a clinical study of chronic hepatitis B patients. The assay was linear in a dynamic 10–10,000 ng/mL range (r²> 0.999) and total analysis time was 6 min in this method. The validated method was applied to quantitatively determine plasma concentration in chronic hepatitis B patients during long‐term telbivudine treatment. The results revealed that telbivudine concentration in the creatine kinase‐elevated group (707.92–2788.78 ng/mL) was significantly higher than those with normal creatine kinase (412.63–1108.32 ng/mL). This method was adapted for therapeutic drug monitoring.     

NMR metabolic profiling of organic and aqueous sea bass extracts: implications in the discrimination of wild and cultured sea bass

The nuclear magnetic resonance (NMR) technique was used as analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples. Preliminary results show that discrimination between wild and cultured sea bass was obtained not only using fatty acid composition but also cholesterol and phosphatidylethanolamine and some water-soluble metabolites such as choline, trimethylamine oxide, glutamine, fumaric and malic acids.     

Microcalorimetric Investigation of Toxic Effects of Three Injectable Solubilizing Excipients on Tetrahymena thermophila BF5 Growth

Using microcalorimetry, thermal metabolic curves of Tetrahymena thermophila BF₅ (T. thermophila BF₅) growth at 28°C affected by three injectable solubilizing excipients (ISE) including tween 80, hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) and poloxamer 188 were measured. Meanwhile, the toxicities of three ISE were evaluated by dynamic parameters of thermal metabolic curves. In addition, the irritative effects of the ISE on myoblast L₆ cells were investigated to show their cytotoxicities by biochemical method. The results indicated that the effects of the ISE on T. thermophila BF₅ varied for different ISE. 5% inhibition concentration values (IC₅) of the ISE were 1.33, 1.83 and 1.64 mg/mL for tween 80, HP‐β‐CD and poloxamer 188, respectively. By the principal component analysis (PCA), the total quantity of heat (Q), growth rate constant (k) and second maximum power (P₂) were selected as the main characteristic parameters to present their toxicities, there were good linear relationships between Q, k, P₂ and concentrations c, suggesting that the toxicities of the ISE on T. thermophila BF₅ were closely linked to their concentrations. The results of creatine kinase (CK) bioassay of myoblast L₆ cells indicated that the sequence of irritative effects of the ISE was HP‐β‐CD相似文献   

High-performance liquid chromatographic determination of creatine kinase activity influenced by methylglyoxal

Protein glycation has been implicated in the development of diabetic complications and other health disorders, which mainly arise from accumulation of advanced glycation endproducts (AGEs) in vivo. Methylglyoxal (MGO), a typical reactive intermediate carbonyl formed in early glycation process, can react non-enzymatically with N-terminal amino groups on proteins, leading to their inactivation and generation of detrimental AGEs. Recently, it was reported that activity of creatine kinase (CK, EC 2.7.3.2) could be reduced or even eliminated completely after incubation with MGO in vitro. CK activity is usually determined by conventional colorimetric assays. However, these methods are not appropriate for monitoring the influence of MGO on CK activity since MGO can also directly react with creatine, a substrate of CK. In this study, an efficient and much more accurate HPLC approach was established to investigate the effect of MGO on CK activity. Aminoguanidine was utilized to eliminate interference from the undesirable reaction between residual MGO and creatine. It was found that higher concentrations of MGO and longer incubation time for CK and MGO caused more pronounced reduction in CK activity. This HPLC method greatly facilitates acquisition of kinetic data about CK reaction and through further improvement it may be adopted to rapidly screen potential inhibitors of MGO-induced glycation.     

Copper(I)–creatine complex on magnetic nanoparticles as a green catalyst for N- and O-arylation in deep eutectic solvent

Immobilization of copper(I) ions on magnetic nanoparticles was performed using surface modification of Fe₃O₄ with creatine. Fe₃O₄@creatine-Cu(I) magnetic catalyst was synthesized and applied in C&bond;X cross-coupling reactions with aryl halides in a deep eutectic as a green solvent. The results indicate the Fe₃O₄@creatine-Cu(I) magnetic nanoparticles showed excellent activity and high stability. In addition, it was revealed that this catalyst can be recycled five times without significant loss in catalytic activity.