Noninterference of Phosphate in an Ethylenediamine Tetraacetate Method for Serum Calcium ALEXANDER D. KENNY and SVEIN U. TOVERUD Biological Research Laboratories, Harvard School o f Dental Medicine, Boston 15, Mass.

S THE determination of calcium in leaf tissue by titration I with the disodium salt of ethylenediaminetetraacetic acid (LDTA), using ammonium purpurate as indicator, Willson (6) encountered interference by orthophosphate when the ratio of phosphorus to calcium was 0.1 or greater (by weight). In cow’s milk, where the ratio of phosphorus to calcium is approximately 0.8, Jenness ( 4 ) overcame the interference of phosphate by remov- ing it with an anion exchange resin.

The authors wished to determine the calcium concentration of rat serum in which, under certain experimental conditions, the phosphorus-calcium ratio may be as high as 4. The ethyl- enediamine tetraacetate method of Fales (I) , with minor modifica- tions, was chosen for this purpose because it requires only 0.5 ml. of serum for each determination rather than 2 ml., as in the classical method of Fiske and Logan ( 2 ) previously in routine use in this laboratory. An investigation of the extent of the interference of phosphate in the new method was considered to be necessary. It has been found that under the specified conditions of the analytical procedure there is no interference even when phosphorus-calcium ratios are as high as 8.

EQUIPMENT, REAGEYTS, AND PROCEDURE

The recommendations of Falep ( 1 ) were followed wtih minor ruceptions. (Details of the modified procedure will be supplied on request.) Microburets (5-ml. capacity, 0.01-ml. graduations) were used instead of pipets for addition of reagents. Aliquots of 0.5 or 1.0 ml. were diluted to 6.0 ml. with distilled water and 0.5 ml. of indicator, freshly filtered, was added. In addition, the following standard solutions were prepared:

Calcium acetate monohydrate (0.44 gram), analytical reagent, was dissolved in water and diluted to 1 liter. The calcium content was found by 14 deter- minations in duplicate by the method of Fiske and Logan ( 2 ) .

Standard Calcium Solution.

Table I . Effect of Phosphate on Analysis of Aqueous Solu- tions and of Serum for Calcium by Ethylenediaminetetra-

acetic Acid P-Ca Ratio - Calcium, JIg. 1100 M I . 0

Solutions Studied Calcd.. Theoryb Found Cxlciiim acetatec 0

0 26 0 .53 0 . 7 8 1.03 1.28 1 .58

8 4 5 8 4,5 8 4.5 8 4 5 8 45 8 4 5 8 45

8.46 8 .41 8.27 8 43 8 . 4 1 8 . 4 8 8.27

Human seruind 0.41 6 14 6.14 1 .51 6 .14 6 . 3 7 2 . 7 1 fi.14 6.19 3.78 6 .14 6.32

Rat serumd 0.59 4.70 4.70 1.65 4 70 4 . 7 1 3.76 4 .70 4.74 5 .84 4.70 4 .76 8 .09 4.70 4.67

a Concentrations are given for solutions analyzed; 0.5-ml. aliquots of cal- cium acetate, calcium chloride, and human serum, and 1.0-ml. aliquots of rat serum were taken.

b On basis of analysis with no added phosphate. C Calcium acetate solution standardized by 14 determinations in duplicate

by method of Fiske and Logan (9). d Phosphorus determined by method of Fiske and Subbarow (3).

Standard Phosphate Solution. iinhydrous potassium dihydro- gen phosphate (4.39 grams), analytical reagent, was dissolved in vvater and diluted to 1 liter after the addition of 10 ml. of lollr sulfuric acid.

Calcium Chloride Solution. .4n aqueous solution containing approximately i . 5 mg. of calcium per 100 ml.

All solutions were prepared with distilled water which contained only negligible amounts of calcium.

RESULTS

Varying amounts of standard phosphate solution were added to standard calcium acetate solution, calcium chloride solution, human and rat blood serum, respectively, to provide a range of phosphorus-calcium ratios which includes values far in excess of expectation in the analysis of serum calcium. 4s shown in Table I, on analysis of aliquots of these solutions for calcium by the present method no evidence of interference by phosphate was disclosed.

DISCUSSIOh

The method here described for analysis of calcium by titration with ethylenediaminetetraacetic acid in a photoelectric color- imeter is applicable to solutions of calcium salts-for example, diluted blood serum-which are far more dilute than the samples tested by Willson and required by his method, which relies upon natural vision for determination of the end point. The calcium concentrations in the solutions titrated (the 1.0-ml. aliquot for human serum and 0.5-ml. aliquots for the other samples were diluted to a total of 6.5 nil. for titration) ranged from 0 42 to 0.72 mg. per 100 ml. as compared with a range of 0.2 to 15 mg. per 100 ml. used by Willson. The authors attribute the absence of interference by phosphate in the present method to the fact that the ion products are well below the solubility product ( K 8 ) of calcium diphosphate (CaHPO,), in contrart to the ronditions of Willson’s experiments.

Difficulties may be encountered in the application of the present method to the analysis of urine, particularly of pathological origin, where phosphorus-calcium ratios as high as 250 are not unknown. Here the prior removal of phosphate with an anion exchange resin, as recommended by Jenness ( 4 ) in another application, may he helpful.

ACKNOWLEDGMEYT

The authors wish to thank F. W. Fales of the Department of Biochemistry, Emory University, for kindly making available the drtails of his method prior to publication.

LITERATURE CITED

(1) Fales, F. W., J . B id . Chem., 204, 577 (1953). (2) Fiske, C . H., and Logan, AI. il., Ibid., 94, 211 (1931) . (3) Fiske, C . H., and Subbarow, Y., Ibid., 66, 375 (1925). (4) Jenness, R . , ANAL. CHEM., 25, 966 (1953). (5) Willson, A. E., Ibid., 22, 1571 (1950) .

RECEIVED for review November 5 , 1953. Accepted February 13, 1954. This investigation was supported in part by a grant-in-aid from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council.

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