Calcium determination in serum with stable alkaline Arsenazo III and triglyceride clearing

  • Morgan B
  • Artiss J
  • Zak B
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We describe an analytical procedure for determining serum calcium, using the ligand Arsenazo III in an aque-ous alkaline medium. The choice of pH for the proposed technique differs from current procedures, which are for the most part carried out in a slightly acidic medium. An acidic medium avoids interference from magnesium, but is spectrophotometrically suboptimal for this pH-depen-dent reaction: the molar absorptivity of the Arsenazo Ill complex with calcium at acid pH is 13 787 L moI1 cm1, about one-half of that at a more optimal alkaline pH (26 574 L mol1 cm-1). We have included a clearing technique in the reagent to avoid spectral aberrations from hypertriglyceridemic samples. a-Cyclodextrin ab-sorbs the nonesterified fatty acids liberated from triglyc-erides by a microbial lipase. This modificationmay also be helpful for binding nonesterifled fatty acids, which are known to interfere with calcium procedures by forming calcium soaps and thus preventing the reaction with intended ligands. The use of 8-hydroxyquinolinesulfonate as the magnesium-masking agent facilitates the use of alkaline pH. The less-water-soluble alternative, 8-hy-droxyquinoline, commonly used as a precipitating agent for several methods, is difficultto solubilize in the alkaline reagent and tends to precipitate when complexed to magnesium. Finally, the use of alkaline pH results tn a prolonged ((6 weeks) shelf life for the reagent. IndexIng Terms: optimization of assay pH . cyclodextrin -ilpase 8-hydroxyquinoilnesuffonate magnesium . imetiy Arsenazo III (ASA III) has been used as a selective ligand for the colorinietric determination of several metals (1-4). However, in the clinical laboratory, its most common application has been in the determination of serum calcium. Since the reported use of ASA III by Michaylova and Ilkova (1) as a reagent for calcium in magnetite, colorimetric calcium determinations modi-fied for serum have been almost exclusively performed at an acidic pH because of the greater selectivity of ASA III for calcium over magnesium. The latter is a major interfering ion of serum in calcium assays that involve alkAline reactions (5). Conversely, the acidic pH is near the isoelectric point of serum proteins, so the risk of protein precipitation, especially with (e.g.) myelomas, is Fax 313-577-0057. 'Present address: Cane Clinic Associates, Department of Pa-thology, Champaign, IL 61801. 2To whom correspondence should be addressed. 3Nonstandard abbreviations: ASA HI, Arsenazo Ill; 8HQ, 8-hy-droxyquinoline; and 8HQS, 8-hyoxyquunoline-5-sulfonate. diminished at a more alkaline pH. We found it of interest that the alkaline region has been almost totally ignored, even though the molar absorptivity of ASA ifi increases with pH, the maximum value occurring at pH 9(1). Therefore, we would think that the conventionally used acidic pH is spectrophotometrically suboptinial for obtaining maximum sensitivity from ASA Ill, even though competition from magnesium is minimized there. 8-Hydroxyquinoline (8HQ) is commonly used as a masking agent for magnesium in methylthymol blue and o-cresolphthalein complexone-based calcium re-agents (5, 6), both carried out in alkaline matrices of reaction. Unlike with both of the previous methodolo-gies, to date there have apparently been few attempts to use ASA III to assay calcium at an alkaline pH, while masking magnesium with 8HQ. However, one early study (7) provided an indirect method for determining calcium by reacting calcium and magnesium with ASA

Author-supplied keywords

  • 8-hydroxyquinoline sulfonate
  • Colorimetry
  • Cyclodextrin
  • Lipase
  • Magnesium
  • Optimization of assay pH

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  • ISSN: 00099147
  • PUI: 23967764
  • SGR: 0027181675
  • PMID: 8353945
  • SCOPUS: 2-s2.0-0027181675


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