Creatine, determination

For creatine determination the appropriate bienzyme electrode was used. Creatinine and creatine could be assayed in parallel by combining a trienzyme electrode and a bienzyme electrode. Both electrodes reached a steady signal 2 min after sample addition. With a 25 pi sample volume the linear range was 1-100 mg/1. The sensors were stable for more than 500 measurements. The CV of creatine determination in serum was 1.3% with 21.7 mg/1 and 11.7% with 8.2 mg/1, and a day-to-day CV of 8.4% was achieved. Comparison with the Jaffe method for 55 serum... 

Description of Method. Creatine is an organic acid found in muscle tissue that supplies energy for muscle contractions. One of its metabolic products is creatinine, which is excreted in urine. Because the concentration of creatinine in urine and serum is an important indication of renal function, rapid methods for its analysis are clinically important. In this method the rate of reaction between creatinine and picrate in an alkaline medium is used to determine the concentration of creatinine in urine. Under the conditions of the analysis, the reaction is first-order in picrate, creatinine, and hydroxide. 

The loss of NADH is followed for determination of the en2yme creatine kinase. 

The overall direction of the reaction will be determined by the relative concentrations of ATP, ADP, Cr, and CrP and the equilibrium constant for the reaction. The enzyme can be considered to have two sites for substrate (or product) binding an adenine nucleotide site, where ATP or ADP binds, and a creatine site, where Cr or CrP is bound. In such a mechanism, ATP and ADP compete for binding at their unique site, while Cr and CrP compete at the specific Cr-, CrP-binding site. Note that no modified enzyme form (E ), such as an E-PO4 intermediate, appears here. The reaction is characterized by rapid and reversible binary ES complex formation, followed by addition of the remaining substrate, and the rate-determining reaction taking place within the ternary complex. 

The effect of a statin is usually determined by measuring fasting plasma lipids and lipoproteins after 4-6 weeks of treatment. Liver enzymes and eventually creatine kinase (in case of myositis liver enzymes are usually also elevated) are measured simultaneously to exclude side effects related to liver and muscles. After the treatment goal has been reached, blood sampling is usually performed 1-2 times a year. 

New manual and automated method for determining activity of creatine kinase isoenzyme MB by use of dithiothreitol Clinical applications. Clin. Chem. (1975), 1612-1618. 

Jost, C. R., Van Der Zee, C. E., In t Zandt, H. J. etal. Creatine kinase B-driven energy transfer in the brain is important for habituation and spatial learning behaviour, mossy fibre field size and determination of seizure susceptibility. Eur. J. Neurosci. 15 692-706, 2002. 

Niacin requires baseline tests of liver function (alanine aminotransferase), uric acid, and glucose. Repeat tests are appropriate at doses of 1,000 to 1,500 mg/day. Symptoms of myopathy or diabetes should be investigated and may require creatine kinase or glucose determinations. Patients with diabetes may require more frequent monitoring. 

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

Immunohistochemical study ND not determined GLUT1 facilitative glucose transporter MCT1 monocarboxylate transporter CRT creatine transporter LAT1 L-type amino acid transporter TAUT taurine transporter ENT equilibrative nucleoside transporter Oatp organic anion-transporting polypeptide PAH p-aminohippuric acid RUI retinal uptake index TR-iBRB rat retinal capillary endothelial cells. 

The strategies for determination of absolute coneentration of metabolites using an internal reference are similar to those of external references. Often tissue water or a metabolite which is expected to have a stable concentration in tissue is used. Total creatine and water are metabolites with relatively stable concentration in musculature, although changes in their rotational mobility may change their NMR visibility. Using water as an internal reference, Eq. (5.1) has to by modified by an additional correction factor according to... 

The isotope tracer method for estimating the total muscle mass of an organism by determining the amount of creatinine clearance. The method relies on the fact that muscle creatine phosphate is converted to creatine, and the latter is metabolized into creatinine. 

In an early application, an enzyme electrode system was reported for the determination of creatinine and creatine, using a combination of creatinine amidohy-drolase, creatine amidinohydrolase and sarcosine oxidase, co-immobilized on an asymmetric cellulose acetate membrane. Thus, the hydrogen peroxide produced was detected to give a quantitative measure of creatine and creatinine in biological fluids [70]. 

Enzymes activities are particularly sensitive to the anticoagulant used in collecting the specimen. Heparin inhibits acid phosphatase (W16) and muramidase (Z5). Amylase activity is inhibited by oxalate or citrate (MIO), and lactic dehydrogenase and acid phosphatase lose activity in oxalate (C2). Alkaline phosphatase is stable in oxalate, oxalate-fluoride, or heparin, but 25 mAf citrate inhibits 50% of the activity, and as little as 50 mlf EDTA is completely inhibitory (B19). Leucine aminopeptidase is inhibited by EDTA, as is creatine phosphokinase (F3). Amylase activity has been reported to be only 83% of that in serum when oxalate or citrate-plasma is used (MIO). Heparin plasma appears to have no inhibitory effect. Despite the fact that clotting factor V is not stable in oxalate or EDTA, these are often used as anticoagulants to obtain plasma for prothrombin determinations (Z2, Z4). 

IM use May increase creatine phosphokinase levels. Use of the enzyme determination without isoenzyme separation, as a diagnostic test for acute Ml, may be compromised. 

The hrefly Inciferin system is very sensitive and can be conpled to any enzymatic reaction that prodnces or nses ATP. For example, creatine phosphokinase can be determined by this method and hence be nsed in the diagnosis of myocardial infarction and mnscle disorders. The creatine phosphokinase converts AMP into ATP which then nndergoes the reaction with Inciferin as shown in Fignre 3.25. ATP pro-dnction is essential for every known life form and the firefly Inciferin system can be nsed to check for microbial life. Hence systems have been developed that use a portable luminescence workstation to monitor sanitation in food manufacturing and to check for sterile environments in technological workplaces. The system can also be applied in checking cell viability, for instance in cell cultures and to measure the toxic effects of chemicals on cells. 

Exploration of Bulk Tolerance. Most affinity labels contain functional groups added to the substrate s basic structure. Discerning just where added bulk can be tolerated by the enzyme is therefore crucial information. In the case of creatine, it has been determined (92,93) that the structures below, for example, are good substitutes for creatine in the creatine-kinase reaction... 

Cognat S, Cheillan D, Piraud M, Roos B, Jakobs C, Vianey-Saban C (2004) Determination of guanidinoacetate and creatine in urine and plasma by liquid chromatography-tandem mass spectrometry. Clin Chem 50 1459-1461... 

Carducci C, Santagata S, Leuzzi V, Carducci C, Artiola C, Giovanniello T, Battini R, Antonozzi I (2006) Quantitative determination of guanidinoacetate and creatine in dried blood spot by flow injection analysis-electrospray tandem mass spectrometry. Clin Chim Acta 364 180-187... 

Reasons for the presence of enzymes in the plasma Enzymes can normally be found in the plasma either because they were specifically secreted to fulfill a function in the blood, or because they were released by dead or damaged cells. Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. The activities of many of these enzymes (for example, creatine kinase, lactate dehydrogenase, and alanine aminotransferase) are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues. 

IR spectroscopy is useful in biochemical research and has aided in the structural determinations of amino acids, nucleic acids, and polypeptides the IR spectra of viruses and bacteria have been studied.9 The controversial cancer drug Krebiozen was identified by its IR spectrum as the amino acid derivative creatine, which is present in humans in large quantities.10... 

The equilibrium constant for the following reaction, which is catalyzed by creatine kinase, has been determined by chemical analysis. The data are given below. [S. A. Kuby and E. A. Noltman, in The Enzymes, 2nd ed. (P. D. Boyer, H. Lardy, and K. Myrback, eds), Vol. VI, pp. 515-602. Academic Press, New York, 1962]... 

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