Colorimetric Estimation of Ultramicro Quantities of Calcium in Human Serum as the Complex with Alizarin SAMUEL NATELSON and RALPH PENNIALL' Department of Biochemisty, Rockford M e m o r i a l Hospital, Rockford,
A rapid colorimetric method suitable for the routine clinical laboratory is described for estimating calcium by extraction of the calcium alizarin complex in n-octyl alcohol. Practical or technical grades of triethanolamine are used to adjust to alkaline pH so as to avoid turbidity in the alcohol phase. Purer grades of triethanolamine are not satisfactory for this purpose. With inorganic alkali iron interference is noted but magnesium, barium, and strontium do not interfere. With organic bases iron does not interfere but interference is noted from magnesium, strontium, and barium. The procedure described for human serum subtracts a constant factor for magnesium interference which is of the order of 7%. The calcium may be precipitated as the oxalate and then determined colorimetrically as the calcium alizarinate in n-octyl alcohol.
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H E R E is a need in the clinical laboratory for a rapid, simple method for calcium estimation on fingertip blood. The method should analyze from 0.01 to 0.02 ml. of serum or 1 to 2 y of calcium. In the search for such a method, a number of procedures which are in use for larger quantities of calcium were investigated. Methods studied include analysis of precipitated calcium oxalate oxidimetrically ( 2 , 5, 33, 36, 38),acidimetrically (34),colorimetrically ( 3 7 ) , and by ethylenediaminetetraacetic acid titration (9, li, 19). Precipitation as the molybdate (16, 39), tungstate (12, 18, 24, 27), phosphate ( 2 5 ) , and sulfate (S), and direct titration with sodium ethylenediaminetetraacetic acid in the presence of Eriochrome Black T (32) or murexide ( 7 , 8 , 10, 21-83, 28, 40) were also studied. Colorimetric estimation with 1,8-dihydroxy-2-(3-chloro-6-hydroxybenzeneazo) naphthalene3,6-disulfonic acid (4)and calcium analysis by flame photometry ( I S , 17, 30, 4 1 ) were also investigated. These methods were found unsuited to the purpose of the present work because of one or more undesirable features, such as the need for too large a sample, long or tedious procedure, or interference caused by icteric or even slightly hemolyzed serum or magnesium. .4lizarin and related compounds were then investigated, since these compounds form intensely colored compleves with calcium as evidenced by their use in staining bone (6) Further, calcium had been assayed by precipitating the oxalate, dissolving in acid, and reprecipitating with alizarin and dissolving the precipitate to read the alizarin colorimetrically (i4). A method was finally devised which was suitable for analyzing 1 to 2 y of calcium rapidly from serum.
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A 4REAGENT ~ (4 nig. ~ per 100 ml,). ~ Alizarin ~ (Eastman ~ I
