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AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 31, No. 4, April, 1959, pp. 314-315 Printed in U.S.A. MG 2 8 STUDIES OF T H E MOLYBDIVANADATE METHOD FOR MAGNESIUM JERRY K. AIKAWA, M.D., AND ELOISE L. RHOADES, B.S. Department of Medicine, University of Colorado School of Medicine, Denver, Colorado In the course of our studies of the metabolism of magnesium with a radioactive isotope of magnesium (Mg28), we needed a rapid but reliable method for the determination of magnesium in biologic fluids and materials. A survey of the various methods currently in use suggested that the method of Simonsen and co-workers1 might be most suitable for our purpose. In this method, calcium is separated from magnesium by precipitation as the oxalate and the magnesium in the supernatant fluid is precipitated as magnesium ammonium phosphate. The concentration of phosphate is determined colorimetrically with molybdivanadate. The purpose of this report is to summarize the results of our studies in which Mg28 was used as a tracer to modify and shorten the method of Simonsen and co-workers.1 MATERIAL AND METHODS The reagents used for the precipitation of calcium and magnesium were those recommended by Simonsen and her associates.1 Mg28 was received as an acid solution of MgCl2. It was neutralized with 1 N NaOH, and the precipitate of Mg(OH) 2 was dissolved in 1 N H2SO4, in order to make a dilute solution of MgS0 4 in distilled water. Mg28 was assayed with a well scintillation counter and a scaling circuit. All countReceived, November 15, 1958; accepted for publication December 19. Dr. Aikawa is Established Investigator of the American Heart Association, and Miss Rhoades is a Research Associate. This work was supported in part by a grant-inaid from the American Heart Association and the Colorado Heart Association and in part by Contract No. AT(ll-l)-282 between the University of Colorado and the U. S. Atomic Energy Commission. Mg28 was supplied by the Brookhaven National Laboratory, Upton, Long Island, New York, on allocation from the U. S. Atomic Energy Commission. ing rates were corrected for physical decay of the isotope. RESULTS Preliminary studies in this laboratory revealed that determinations of magnesium on duplicate and triplicate samples of pooled serums performed on different days according to the original method of Simonsen and her associates1 agreed within 2.0 =fc 3.7 per cent of the over-all mean value in 11 separate runs. Triplicate determinations of the phosphate standard on 12 occasions were reproducible within 1.2 ± 1.5 per cent. When known amounts of magnesium were added to pooled serums, recovery of the added magnesium was complete. Mg-S Studies with Serum Tracer quantities of Mg28 were added to pooled serums in order to follow stepwise the completeness of separation of magnesium from calcium. Whether the calcium oxalate precipitate was allowed to form for 5, 10, 15, 30, 60, 90, or 120 min., or 24 hr., a mean of 2.98 per cent of the radioactivity was recovered with the precipitate after the supernatant fluid was carefully decanted off. The supernatant fluid contained a mean of 96.80 per cent of the radioactivity originally present in the mixture. Two washings of the oxalate precipitate with 2 per cent NH 4 OH removed all but 0.16 per cent of the radioactivity. The minimal time interval necessary for the complete precipitation of calcium from the serum as calcium oxalate was next determined. Precipitation for 5, 10, 15, 20, 25, 30, 60, 90, or 120 min. resulted in the same amount of calcium in the washed precipitate, as determined by titration of permanganate. The minimal time interval necessary for the complete precipitation from serum of magnesium as magnesium ammonium phosphate was determined as follows: The super- 314 METHOD FOR MAGNESIUM April 1959 imtant fluid above the MgNH 4 P0 4 precipitate, allowed to form for 5, 10, and 15 min., contained up to 6.70 per cent of the radioactivity. When precipitation for 30 min. or longer was allowed, the supernatant fluid contained a negligible amount of radioactivity. After precipitation for 30 min., 2 washings of the MgNH 4 P04 precipitate with alcoholic ammonium hydroxide resulted in a mean decrease of 1.90 per cent in the radioactivity content of the precipitate. Thus, a mean of 94.90 per cent of the original Mg28 was recovered in the final precipitate. The phosphate content of the MgNH 4 P04 precipitate at 5, 10, 15, 20, 25, and 30 min. manifested a progressive increase with the time allowed for precipitation. After the precipitate had been allowed to form for 30, 60, 90, and 120 min., the concentrations of phosphate of the precipitate were identical. Mg28 Studies with Urine A tracer amount of Mg28 was added to a pooled specimen of human urine and the fate of the radioactivity followed after the precipitation of calcium for 10 min. and the magnesium as phosphate for 30 min. A mean of 2.17 per cent of the Mg28 was recovered with the calcium oxalate precipitate and a mean of 99.00 per cent was found in the washed MgNH 4 P0 4 precipitate. COMMENTS Precipitation of calcium as calcium oxalate was complete within 5 min. Precipitation of magnesium as magnesium ammonium phosphate was complete by 30 min. It was essential that the mixture be stirred vigorously for at least 1 min. after the addition of (NH 4 ) 3 P0 4 . With these reductions in 315 time intervals for precipitation, the method becomes rapid and suitable for clinical use. Concentrations of Magnesium in Serum from Human Beings Two hundred and twenty determinations of concentrations of magnesium in serum of hospitalized persons by this modified method revealed a mean value of 1.59 ± 0.32 mEq. per 1. Values lower than 1.10 mEq. per 1. were obtained in serums from patients with congestive heart failure, cirrhosis, and renal failure after dialysis. All values higher than 2.10 mEq. per 1. were found in patients with renal failure prior to therapy. SUMMARY Mg28 was used as a tracer to study the characteristics of the method of Simonsen and her associates for the determination of magnesium in serum and urine. Precipitation of calcium was complete within 5 min. and the adsorption of magnesium to this precipitate was minimal. Precipitation of magnesium as magnesium ammonium phosphate was complete by 30 min. SUMMARIO IN INTERLINGUA 28 Mg esseva usate como traciator in le studio del characteristicas del methodo de Simonsen e associatos pro determinar magnesium in sero e urina. Le precipitation de calcium esseva complete intra 5 minutas. Le adsorption de magnesium a iste precipitate esseva minimal. Le precipitation de magnesium in le forma de phosphate ammoniaco-magnesian esseva complete in 30 minutas. REFERENCE 1. SIMONSEN, D. G., WESTOVBR, L. M., AND WERTMAN, M.: The determination of serum magnesium bv the molvbdivanadate method for phosphate. J. Biol. Chem., 169: 39-47, 1947.