A METHOD FOR THE COLORIMETRIC DETERMINATION OF

CALCIUM AND MAGNESIUM IN SMALL AMOUNTS OF

URINE, STOOL, AND FOOD*
BY GEORGE D. MICHAELS, CARL T. ANDERSON, SHELDON MARGEN,
AND LAURANCE W. KINSELL
(From the Division of Medicine, University of California Medical School, and the
Metabolic Research Unit, United States Naval Hospital-University of California
Medical School, Oakland and San Francisco)
(Received for publication, April 4, 1949)

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This report presents a method for the quantitative determination of
magnesium and calcium in a single specimen of urine, stool, and food by
formation and calorimetric determination of their respective phosphates.
The method makes use of someof the principles advanced by Roe and Kahn
(1) for determination of serum calcium, and by Denis (2) for serum mag-
nesium. Phosphorus determinations are performed by the method of
Fiske and Subbarow (3).
Principle
The principle involved relates to the conversion of magnesium and cal-
cium to their phosphates; determination of the total phosphate (Mg + Ca);
precipitation of the calcium as oxalate, and of the magnesium (after sepa-
ration) as magnesium ammonium phosphate. The calcium is then redis-
solved and reprecipitated as calcium phosphate. Both phosphates are
then determined, and should equal the total phosphate above noted.
Reagents-
1. Phosphorus reagents (Fiske and Subbarow (3)).
2. 5 per cent ammonium phosphate (secondary) solution.
3. Ammonium hydroxide, c.P., 28 per cent.
4. 20 per cent sodium acetate solution.
5. 2.5 per cent oxalic acid.
6. Alcohol wash containing 25 ml. of 5 per cent ammonium chloride,
100 ml. of ammonium hydroxide, 200 ml. of 95 per cent ethyl alcohol,
15 ml. of amyl alcohol, and 160 ml. of distilled water.
7. 1~4 dilution of concentrated hydrochloric acid.
8. 1:50 dilution of 28 per cent ammonium hydroxide.
9. Indicator (methyl orange, pH range 3.1 to 4.4).
* This work is supported by grants from the Research Division of the Bureau of
Medicine and Surgery, United States Navy (BuMed. No. 007046) and from the Office
of Naval Research, under a contract between the latter and the University of Cali-
fornia
175
176 COLORIMETRIC DETERMINATION OF CA AND MMa

Method
Calcium and Magnesium Analysis of Urine
1. To a 15 ml. conical centrifuge tube add 10 ml. of well shaken urine,
2 drops of methyl orange, concentrated hydrochloric acid dropwise, to
a red color, and 1 ml. of 5 per cent ammonium phosphate slowly with
shaking.
2. Add 2 ml. of ammonium hydroxide (28 per cent), mix, and let stand
for at least 1 hour.
3. Centrifuge, decant, and wash the precipitate three times with 5 ml.
of alcohol wash.

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4. Dissolve the precipitate with 0.5 ml. of I:4 hydrochloric acid and
wash quantitatively into a 10 ml. volumetric flask, bring to volume with
distilled water, and mix.
5. 1 or 2 ml. of this are then used for total phosphate determination.
6. 5 ml. of the aliquot from item (4) are taken and 1 ml. of 2.5 per cent
oxalic acid is added, as well as a drop of methyl orange. Sodium acetate
solution is then added slowly until the pH is approximately 4.0; i.e., until
the indicator just turns from red to orange. The mixture is then allowed
to stand for 4 hours or more for the complete precipitation of the calcium
oxalate. This assures complete calcium precipitation and magnesium
solution (pH below 5.0 and above 2.5), as shown by Washburn and Shear
(4).
7. The precipitate of calcium ox&late is then centrifuged and washed
twice with 3 ml. of 2 per cent ammonium hydroxide. The supernatant and
the washings are saved for the magnesium determination.
8. The calcium oxalate precipitate is dissolved in 0.5 ml. of 1:4 hydro-
chloric acid and 5 ml. of water. 0.5 ml. of 5 per cent ammonium phosphate
and 2 ml. of ammonium hydroxide are added and mixed to reprecipitate
the calcium as the phosphate. After allowing this to stand for 1 hour or
more, it is centrifuged and the precipitate washed twice with the alcohol
wash. (The 40 per cent alcohol wash will not precipitate phosphates of
sodium, potassium, and ammonium.) The precipitate is then dissolved
with 0.5 ml. of 1:4 hydrochloric acid and the entire amount or a portion
may be used for the phosphorus determination, depending upon t,he
amount of calcium present.
9. To the supernatant and the washing from the above, 0.5 ml. of
ammonium phosphate solution and 2.0 ml. of strong ammonia are added
and allowed to stand for 1 hour or longer. This precipitate (magnesium
ammonium phosphate) is centrifuged and washed twice with 5 ml. of
alcohol wash and then redissolved in 1:4 hydrochloric acid. Phosphate
analysis is then performed upon (1) the total ph0sphat.e from step (5),
 MMICHAELS, U’DERSON, MARGEN, #ND KINSELL 177

(2) the calcium‘phosphate from step (S), (3) the magnesium ammonium
phosphate from step (9).
Actual phosphate determinations are performed by the method of Fiske
and Subbarow, as previously noted. Colorimetry is performed with any

TABLE I
Calcium-Phosphorus and Magnesium-Phosphorus Ratios in Aqueous Solutions
Containing Known Amounts of Calcium
-7
Specimen No. Ca added P recovered Ca:P Mg added P recovered Mg:P
___. --
w. nrg.

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0.273 0:;67 1.63 or”512 0.654 0.783
0.273 0.168 1.62 0.512 0.654 0.783
0.546 0.336 1.62 1.025 1.293 0.792
0.546 0.336 1.62 1.025 1.308 0.783
0.819 0.501 1.63 1.536 1.982 0.775
0.819 0.504 1.62 1.536 1.962 0.783
3.075 3.914 0.785
3.075 3.974 0.774
- --
Average .. ... ... ... . 1.62 0.782
-

TABLE II
Predicted and Actual Phosphorus Recovery in Aqueous Solutions, Containing
Known Amounts of Calcium and Magnesium
With the factors noted in Table I, excellent correspondence is obtained between
predicted and actual values.
-
7
Specimen No. Ca added I@ added Per cent
recovery

0.821 oY20 100.0

0.273 0.512 0.821 0.815 99.3
0.546 1.025 1.642 1.631 99.3
0.546 1.025 1.642 ’ 1.639 99.8
0.819 1.536 2.463 2.453 99.6
0.819 1.536 2.463 2.458 99.8

standard photoelectric calorimeter, having a green filter with an approxi-

mate spectral range of 500 to 570 rnp.
CaEcium and Magnesium
Analysis of Food and Stool
Stool and food are dry-ashed for a minimum of 12 hours at a tempera-
ture of NO-600’.
178 COLORIMETRIC DETERMINATION OF CA AND MC

The amount of ash derived from 2 gm. of dry stool or food is dissolved
in 5 ml. of warm 1:4 hydrochloric acid. This solution is made up to 50
ml. with distilled water in a volumetric flask. The procedure thereafter
is as described for urine, except that the size of the aliquot will vary with
the calcium and magnesium concentration. Best results are obtained with
a final phosphorus concentration of 0.3 to 0.8 mg. per 100 ml.
Calculation (See Below for Derivation)-Ng. of calcium = mg. of phos-
phorus X factor 1.62; mg. of magnesium = mg. of phosphorus X factor
0.782.
Analysis 01Known Calcium-Magnesium fJoluticms

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Since silicates are known to interfere with the phosphorus determination,
we have avoided the use of strong bases in the conversion of calcium

TABLE III
Recovery oj Added Calcium. and A1agnesium from Pooled Urine, by Use of Ca:P and
Mg:P Factors from Table I
--

and magnesium to their phosphates. Ammonium hydroxide, being a

weak base, does not form silicates and does form a compound or compounds
having a constant calcium-phosphorus ratio of 1.62: 1, as shown in Table
I. The magnesium to phosphorus ratio is 0.782: 1.’ This would indicate
t,he formation of magnesium ammonium phosphate, whereas, in the ease
of calcium, apparently an equal mixture of tricalcium phosphate and
ammonium calcium phosphate results. Analysis of known mixtures of
calcium and magnesium in distilled water gave rise to the above ratios.
Recovery of total phosphates ranged from 100 to 99.3 per cent, as shown
in Table II.
A second experiment similar to the one just described was performed
upon previously analyzed pooled urine to which were added known quan-
tities of calcium and magnesium. The data are recorded on Table III.
Calcium and magnesium are recovered quantitatively.
1 The magnesium standard was prepared as described by Jones (5).
 MICHAEL& ANDERSON, MARGEN, AND KINSELL 179

Table IV shows the comparisons of the calcium values on food, urine,

and stools by McCrudden’s method (6) and the one here described.
Considerable variation is noted in some instances. It is worthy of
emphasis that, as shown in Table IV, values obtained by the’method herein
described are reproducible.
To date 209 urinalyses, thirty-two diet analyses, and forty-nine stool
analyses have been performed in duplicate in this laboratory by the method
reported. Low variability is a consistent finding.
TABLE IV
Calcium Determination in Food, Stool, and Urine; Comparison of Two Methods
- - - -

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Specimens McCntdden’s Variation Phosphate Variation
analyzed method, duplicate I nethod, duplicate
analyses Iper sample analyses F ersample
-
%za?:E& w. ?Es % 2: fw.
Food Amigen 0.458 0.440 18 0.444 0.440
Milk 1 0.114 0.112 2 0.102 0.102
(‘ 2 0.118 0.115 3 0.117 0.116
Blended food 0.019 0.019 0 0.021 0.021
Veal 0.017 0.018 1 0.011 0.011
Beef 0.008 0.007 1 0.009 0.009
.cm. gm.
24R”’
IS. 24R”’
IS. %h?:: Fh??
Urine Sm 0.102 0.100 2 0.098 0.097
Pa 0.123 0.122 1 0.125 0.124
MaI 0.074 0.072 2 0.082 0.082
Mat 0.073 0.072 1 0.072 0.072
Stool Sm 0.651 0.623 28 0.676 0.673
Pa 1.866 1.834 32 1.854 1.854
Ma1 1.410 1.410 0 1.370 1.370
Ma? 1.120 1.034 86 1.010 1.002
-

SUMMARY

1. The determination of calcium phosphate, magnesium phosphate, and

combined phosphate, as described, provides a constant check of the accur-
acy of the procedure in terms of loss of material; it does not provide a check
on the magnesium-calcium separation. Therefore, proper control of pH
is essential, as noted above.
2. Initial precipitation of calcium and magnesium in the presence of an
excess of phosphate, and in the absence of strong alkali eliminates inter-
fering 8UbStaCeS such as sulfates and silicates, as suggested by Fiske and
Logan (7) and Roe and Kahn (1).
3. The simplicity and accuracy of the method commend its use, particu-
larly when multiple urine and stool determinations or small amounts of
material are required, as in the case of metabolic studies generally.
I80 COLOBIMETItIC DETERMINATION OF CA AND IvfG

BIBLIOGRAPEIY
1. Roe, J. N., and Kahn, B. S., J. Biol. them., $7, 585 (1926); 81, 1 (1929)
2. Denis, W., J. Biol. Chem., 52, 411 (1922).
3. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 86, 375 (1925).
4. Washburn, M. L., and Shear, M. J., J. Bid. Chem.. 99, 21 (1931-32).
5. Jones, W., J. Biol. Chem., 25, 87 (1916).
6. McCrudden, F. H., J. Biol. Chem., 10, 187 (1911-12).
7. Fiske, C. H., and Logan, M. A., J. Biol. Chem., 93, 211 (1931).

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