Creatine kinase, assay

Urea standard solutions in water 3.57 mM, 8.92 mM, 10.71 mM, and 35.70 mM Creatine kinase assay reagent consisting of ... 

Why was it necessary to know the exact incubation times for the creatine kinase assays per-... 

Methods have recently been used for measuring arm-to-arm (total) circulation time separately (D6) or simultaneously with arm-tongue (central) circulation time (D5), and abnormal reductions in the difier-ence (peripheral circulation time) predictably found in dystrophic patients (D3) have been found also in female carriers of Duchenne-type dystrophy (D5a, S18) and may be due to increased metabolism of the diseased muscle. By combining these techniques with simultaneous serum aldolase and creatine kinase assay, successful detection of 85-90% of carriers is said to be possible (D7, D7a, D8). Further, electromyographic studies in known carriers have now disclosed the presence of polyphasic responses intermediate in frequency between those found in normal and in dystrophic individuals, thus providing an additional means of discrimination (VI). 

Several luminescent systems have been described, one of the more commonly used being the firefly luciferase system. This uses ATP as one of its substrates and light is emitted during the course of the chemical reaction. The enzyme can therefore be used in reactions where ATP is formed or consumed, e.g. creatine kinase assay, or the measurement of glycerol by glycerol kinase. Luminescent immunoassays, analogous to radioimmunoassay or enzyme-immunoassay have also been described, using luminol or luminol derivatives as the label. 

Dinovo, E. C. Miyada, D. S. and Nakamura, R. H. Evaluation of direct and indirect compled enzyme assay systems for measurements of creatine kinase activity. 

Figure 6, High pressure liquid chromatogram of creatine kinase isoenzymes. First peak, MM second peak, BB. Conditions 50 cm X 4.8 mm (i.d.) column with yydac porous layer bead anion exchange mobile phase, step gradient Solvent A, 10 mmol/liter Tris buffer, pH 8.3 solvent B, 10 mmol/liter Tris buffer, pH 7.0,0.5 mol KCl flow rate, 2 ml/min detection, collected fractions assayed (45).

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

Many assays have been described in which the initial product forms the substrate of an intermediary reaction involving auxiliary enzymes. The assay of creatine kinase (EC 2.13.2), for example, involves hexokinase (EC 2.7.1.1) as the auxiliary enzyme and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as the indicator enzyme ... 

Occasionally, one may also wish to use an auxiliary enzyme not as an assay system but strictly as a means for maintaining the steady-state concentration of a primary reactant in a multisubstrate reaction system. For instance, acetate kinase (and its substrate acetyl phosphate) or creatine kinase (and its substrate creatine phos-... 

Cost-selectivity equation 395-399 Coupled assays 196 Covalent catalysis 62, 77-85 see also nucleophilic catalysis Creatine kinase 264, 266 Crotonase 108, 166 Cryoenzymology 40 Crystalline enzymes activity 45 water content 39 Curie 196... 

Creatine kinase (CK) -clinical assay for [AUTOMATED INSTRUMENTATION - CLINICALCHEMISTRY] (Vol3)... 

Plasma CHE was determined by a radiometric assay (9) using acetylcholine and Btf 284c51. Plasma creatine kinase fCK) was determined by a spectrophotometric method (10). 

Scheme 2. Reaction pathways utilising a redox enzyme linked amperometric measurement via a redox mediator, (b) assay for glucose (a)+(b) assay for creatine, creatine kinase or hexokinase (c) competitive immunoassay using Fc-labelled antigen.

Figure 16-3 Assay for serum creatine kinase (CK) activity.

The assays for serum glucose and creatine kinase were similar in many respects. List the chemical reactions that were common to both assays. Also, describe how these two similar assay systems could be used to quantify these two very different biomolecules. 

Fig. 12.35. Quadruple-label immunoassays of four analytes (thyroid stimulating hormone, 17a-hydroxyprogesterone, immunoreactive trypsin and creatine kinase) in a single assay cuvette with...

After the mixture had been allowed to cool, a 10 /uL sample was analyzed by HPLC. The results of one assay are shown in Figure 9.101. The appearance of the ATP is clearly noted and is evidence of creatine kinase activity. 

Creatine kinase is a protein necessary for ATP generation. One of its forms, CK-MB, is found mainly in the myocardium and upon tissue damage, such as myocardial infarction (Ml), becomes elevated. It takes up to 24 h for the elevated level to reach its peak. A difficulty resides in the fact that unlike troponin, an assay does not allow us to distinguish between cardiac and skeletal muscle damage. Also, in about a third of Ml cases while CK-MB levels stay neutral, troponin elevation is noted. The normal level of troponin in the blood is less than 0.3 pg/1 while for CK-MB it is less than 3.0 ng/ml. For humans, the CK is in the range of 55-170 lU/1 (international units per liter) and is less specific than CK-MB for cardiac tissue damage. 

It was soon noted, however, that serum creatine kinase as well as aldolase may be elevated in carriers (S12, S33), that here creatine kinase is far more sensitive, and that by its assay not only might the carrier state be confirmed in almost every female known to harbor the gene (Al, H8), but the expected proportion of mutant cases could be found by a family absence of both clinical and biochemical evidence of heredity (R12). These results have been confirmed for creatine kinase in a very large series of relatives of Duchenne-type dystrophic patients (R4), although... 

During an incubation and measurement phase of altogether 240 seconds, the reflectance values of the formed dye are measured at 340 nm every 5 seconds between the 160th and 240th second. 7V-acetylcysteine was added to activate the oxidized creatine kinase (CK). Myokinase inhibitors prevent interference with the assay by this enzyme. From the measured reflectance values the instrument calculates the CK activity of the sample via a calibration curve produced with two different calibrators (high/low). 

ST53 Butch, A.W., Goodnow, T.T., Brown, W.S., McCellan, A., Kessler, G. and Scott, M.G. (1989). Stratus automated creatine kinase-MB assay evaluated Identification and elimination of falsely increased results associated with a high-molecular-mass form of alkaline phosphatase. Clin. Chem. 35, 2048-2053. 

ST81 Christenson, R.H., Ohman, E.M., Clemmensen, P., Grande, P. and Wagner, G.S. (1990). Assay dependency for assessing creatine kinase-MB release after myocardial reperfusion. Clin. Chem. 36, 1129, Abstr. 827. 

ST92 Lianidou, E.S., Christopoulos, T.K. and Diamandis, E.P. (1990). Assay of creatine kinase isoenzyme MB in serum with time-resolved immunofluorom-etry. Clin. Chem. 36, 1679-1683. 

STN5 Christenson, R.H., Kazanzides, D., Pizzo, C.K. and Nadkami, S. (1991). Verification of plasma based calibrators for use with the Stratus creatine kinase-MB assay. Clin. Chem. 37, 912, Abstr. 15. 

A commercial serum creatine kinase assay8 employs the kinetic method for enzyme quantitation. This three-enzyme, coupled assay involves the following sequence of reactions ... 

Immunoassay kits using luminol and arylacridinium ester labels have been developed by nine companies for a variety of thyroid, steroid and pituitary hormones, viruses, digoxin, and creatine kinase. One assay for total and free thyroxin has detection limits of 20 pM in serum, while another for total and free triiodothyronine has a 3-pM detection limit in serum. These analytes are used as clinical indicators of thyroid gland malfunction. While research has shown that... 

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