BE905345A - Creatine phosphate assay - using creatine kinase and creatinase - Google Patents

Abstract

Determn. of creatine phosphate (CP) in body fluids (plasma, serum, serum ultrafiltrates, urine or tissue extracts) is effected by contacting the sample with creatine kinase (CK), which converts CP to creatine, and with creatinase, which converts the creatine in a detectable reaction as described in BE-904688.

Description

       

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  4. DESCRIPTION OF THE SYSTEM.



   DOSAGE OF CREATIN PHOSPHATE.



  Creatine phosphate dosing is performed on samples of body fluids (plasma, serum and their ultrafiltrates, urine or tissue extracts).



  The analysis is carried out according to an enzymatic method, which uses creatine kinase and creatinase. This reaction is feasible in a liquid medium (aqueous solution) as well as on a solid support.



  In this method, the creatine phosphate is converted into creatine using creatine kinase. The creatine thus formed is further converted via creatinase and a second monitor reaction as described in Belgian Patent 904. 688 (Min. Decided.



  15. 05, 86).



  A parallel determination, this time without adding creatine kinase, but otherwise completely identical to the first determination, serves to determine the blank value.



  The net creatine phosphate content is obtained by subtracting the result of the blank measurement from the first measurement.

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  RECIPE Creatine phosphate is determined by mixing steel and reagent in the ratio 1 (sample) to 20 (reagent).



  The reaction proceeds at temperatures between 15 and 40 ° C.



  The reagent consists of a mixture of 50% Tris buffer pH 7.4; strength: 150 mmol / 1 and 50% Potassium phosphate buffer pH 7.4; strength 150 mmol / l. Deviations from this mixing ratio do not affect the reaction as long as the Tris buffer is between 40
 EMI2.1
 and occupies 95% of the reaction volume.



  ADDITIONS TO THE POTASSIUM PHOSPHATE BUFFER T ') aluminum hexacyanoterrat (II) 2) 2, 3). Sodium cholate 5 mmol / l 4) Triton X-100 0.25%
The following is added to this mixture: 4-aminophenazone 0.8 mol / 1 creatinase 30 U / ml sarcosine oxidase 10 U / ml lipase 2U / ml creatine kinase 29 U / ml
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 ADP-di sodium 0, MgC12 6 H20 7, peroxidase 2 U / ml ascorbate oxidase 20 U / ml

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 5. SITUATION IN THE DOMAIN.



  The determination of creatine phosphate with this method represents a significant improvement over previous methods since: 1. This method is faster than the previously used method: This represents an important advantage when dosing creatine phosphate in poorly stable biological material.



  2. Due to the fully enzymatic character of the method, this method is very specific and easy to measure. After all, the reading takes place at a wavelength between 500 and 600 nm; a spectral zone where few interferences are expected in biological material.


    

Claims (2)

6. APPLICATIONS. 1. Evaluate and establish energy balances in tissues by measuring creatine phosphate concentrations in the above mentioned biological environments. 2. For the diagnosis of skeletal and cardiac muscle necrosis and / or ischemia and follow-up during the early (first day) post-myocardial infarction period by determining the concentrations of creatine phosphate released in the extracellular environment from damaged skeletal muscle or heart muscle cells.  <Desc / Clms Page number 5>    EMI5.1   Drawn up in Ghent on August 28, 1986 DELANGHE Joris DE SCHEERDER r, /, DE BUYZERE M "/ -. \ ''. "GHEERAERT Peter t WIEME Roger