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Enzymatic method for assaying calcium in serum with Ca++ -ATPase
Masood-ul-Hassan Javed1, Francesco Michelangeli
1Department of Biochemistry, The Aga Khan University, Karachi, Pakistan.
Experimental & Molecular Medicine
|March 19, 2003
Summary
A novel kinetic assay accurately measures total serum calcium by monitoring Ca(++)-ATPase activation. This method demonstrates high precision and minimal interference, offering a reliable alternative for clinical diagnostics.
Area of Science:
- Biochemistry
- Clinical Chemistry
- Analytical Chemistry
Background:
- Accurate measurement of total serum calcium is crucial for diagnosing various medical conditions.
- Existing methods may have limitations in precision, interference, or complexity.
Purpose of the Study:
- To develop and validate a new kinetic assay for determining total calcium concentration in human serum.
- To assess the assay's accuracy, precision, and reliability compared to a standard method.
Main Methods:
- A kinetic assay was developed utilizing the activation of Ca(++)-ATPase by free calcium ions (Ca(++)f).
- Free calcium concentration was controlled using EGTA, and Ca(++)-ATPase activity was coupled to NADH reduction via pyruvate kinase and lactate dehydrogenase.
- Absorbance changes at 340 nm were monitored to quantify enzyme activity, reflecting total calcium levels.
Main Results:
- The developed assay yielded a mean total calcium value of 10.08 ± 0.24 mg/dl for normal serum.
- Results showed excellent agreement with the o-cresolphthalein complexone (CPC) method (r = 0.912).
- Within-run coefficients of variation ranged from 0.9-2.87%, and day-to-day variations were 0.72-3.17%, indicating high precision.
Conclusions:
- The developed kinetic assay provides a precise and reliable method for measuring total serum calcium.
- The assay is robust, showing minimal interference from common ions and clinical agents.
- This method offers a valuable alternative for routine clinical laboratory analysis of serum calcium.