Novel methodology for the determination of alkaline phosphatase isoenzymes in plasma circulating

GUGLIELMI, CECILIA; BRUN LUCAS; RIGALLI ALFREDO

Córdoba

Congreso; XXVII Reunión anual de la Asociación Argentina de Osteología y Metabolismo Mineral; 2010

Asociación Argentina de osteología y Metabolismo Mineral

p { margin-bottom: 0.21cm; } Alkaline phosphatase (AP) exist in plasma as isoenzymes: bone (bAP), liver (lAP) and intestinal (iAP).The activity of bAP is particularly useful as a measure of bone formation. The objectives of this work was to characterize the activity of the AP isoenzymes of rat serum on the basis of the sensitivity of temperature (T), pH and concentration of phenylalanine (Phe) and develop a methodology to measureindividual activities in a mixture of them. A mathematical model is proposed, which is based on a set of linear algebraic equations, where the activities of bAP, lAP and iAP in plasma are the unknowns values. The activity of FA and the percentages of activity of each isoenzyme at a given value of T, pH, and Phe are the independent terms and the coefficients, respectively. The activity of each isoenzyme was measured at pH=8, T=37 ºC, Phe=0 mM and varying the incubation medium:a) pH= 6-10, b) T= 10-45 °C; c) Phe= 0-16 mM.The percentages of activity of each isoenzyme for the range of each variable were calculated considering 100% the value of activity measured at pH=8, T=37 ºC and Phe=0mM.In a mixture of the three isoenzymes at a given value, the total activity of the AP is the sum of the activities for each isoenzyme multiply by its percentage of activity. The solution of the system are the values of activities of bAP, lAP and iAP in plasma. The activity of each isoenzyme was measured in an artificial mixture of the three purified isoenzymes (90% FAi, 5% FAo, 5% FAh). The values obtained were: FAi: 3.15 pNFF pmol/L.seg (68.12%); FAo: 0.75 (16.18%) y FAh: 0.73 (15.70%). Although they are preliminary results, the proposed methodology has acceptable values.