IMPROVEMENTS IN THE METHODS FOR CALCIUM DETERMINATION IN BIOLOGICAL MATERIAL

BY CHI CHE WANG

(Prom the Children’s Hospital Research Foundation and the Department of Pediatrics, University of Cincinnati, Cincinnati)

(Received for publication, June 10, 1935)

It is well known that the principal disadvantage of any method for the determination of calcium involving precipitation as cal- cium oxalate lies in washing the precipitate. The solubility of the precipitate in water or in dilute ammonium hydroxide is ap- preciable and, in centrifuging, a small amount of calcium oxalate tends to float on the surface of the liquid and is lost in decanting. The satisfactory results obtained by the oxalate method are due largely to the compensation of errors, the loss of the precipitate of calcium oxalate being balanced by incomplete removal of am- monium oxalate in the washings. It is apparent that a washing fluid which would remove the excess of oxalate solution with no appreciable solution of the precipitate and which prevents any of the precipitate from floating on the surface of the liquid would increase the accuracy of the method. It has been found that a mixture of 2 per cent ammonia in equal parts of alcohol, ether, and distilled water meets these requirements, provided that the ammonium oxalate used for precipitation is not too concentrated. 0.1 M ammonium oxalate is substitutued for the usual saturated ammonium oxalate (4 per cent). This is about one-third the usual concentration but insures an adequate oxalate concentra- tion for complete precipitation of calcium oxalate. Two washings with the ammoniacal alcohol-ether-water mixture are needed but there is no appreciable loss of calcium with three washings. Three washings are not sufficient when more concentrated ammonium oxalate is used.

In applying the new washing solution to calcium determinations we have used Kramer and Tisdall’s (1) permanganate titration,

443

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TABLE I

Calcium Determinations with New Washing Technique

All precipitations stood overnight.

On calcium chloride solutions (Iceland *pm in HCI)

-- c?. ,mg. pm cent mg.

0.224 0.228 +1.8 0.124 0.224 0.227 +1.3 0.124

0.224 0.224 0.0 0.124 0.224 0.227 f1.3 0.124 0.224 0.228 +1.8 0.124 0.224 0.225 +0.4 0.124

__-__ Average.0.224 0.226 +l.l 0.124

Ca deter- mined

ms. per cm1

0.123 -0.8 0.123 -0.8 0.122 -1.6

0.123 -0.8 0.124 0.0

0.124 0.0

0.123 -0.7

--- %?. mg. per cent

0.062 0.061 -1.6 0.062 0.060 -3.2

0.062 0.062 0.0 0.062 0.061 -1.6 0.062 0.062 0.0 0.062 0.063 -1.6

--~ 0.062 0.062 f1.3

Recovery of Ca from artificial tissue extract containing per 100 cc. 8 mg. Ca, 20 mg. Mg, 152 mg. P, 150 mg. Na. and 190 mg. K

Diluted 80 5 cc. sample = 0.080 mg. Ca Diluted 80 5 cc. sample = 0.200 mg. Ca

Pptd. directly

mg.

0.079 0.080 0.078 0.078

0.082 0.078

0.081 0.082

Pptd. directly

w7.

0.200 0.202 0.201 0.198

0.197 0.197

Average.0.080 0.199

Ashed with 5 dro s HzSOa. taken up mt HCl .%

“8.

0.201 0.201 0.197

0.198 0.200 0.201

0.200

Dog 8erum (1 cc;~$valent of serum Recovery of Ca added to dog m m (trichloroacetic acid filtrate)

Ashed with Trichloroacetic acid Cs in serum

filtrate Ell~g%& detgp- Ca *dckJd

w7. mg. m?. “9.

0.099 0.097 0.021 0.062

0.098 0.098 0.069 0.062 0.100 0.100 0.080 0.062

0.101 0.101 0.059 0.124 0.100 0.102 0.060 0.124 0.103 0.099 0.042 0.186 0.097 0.097 0.128 0.124

0.098 0.097

Avcrage.0.100 0.099

444

Added Ca recovered

m7. per cent

0.063 $1.6

0.062 0.0 0.063 f1.6

0.126 f1.6 0.127 +2.4 0.187 $0.5 0.127 +2.4

+1.5

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C. C. Wang 445

Clark and Collip’s (2) method of removing the washing fluid, and Van Slyke and Sendroy’s (3) principle of removing proteins with trichloroacetic acid.

The method, as applied to samples containing between 0.06 and 0.22 mg. of calcium, gave an average deviation of ~1.1 per cent of theoretical values with standard CaC& solution or with artificial tissue extract solution, and an average deviation of $1.5 per cent when calcium was added to the filtrate from blood serum (Table I). After a little experience, one person can easily run twenty-four calcium determinations during a working day. The author has run as many as forty-eight in a day. In connec- tion with our metabolic work, this method has been used by the writer in approximately 2500 determinations of calcium, in blood, urine, food, and feces, with an error of approximately 2 per cent. The procedure also has been successfully used by associates in other departments of this institution. The exact techniques for serum, urine, food, and feces are.given below.

Reagents

Trichloroacetic acid, 20 per cent; 20 gm. dissolved and diluted to 100 cc.

Sodium acetate, 20 per cent; 20 gm. dissolved and diluted to 100 cc.

Brom-cresol green, 0.016 per cent; prepared from stock solution as described by Clark (4).

Ammonium oxalate, 0.1 M; 1.42 gm. of ammonium oxalate dis- solved and diluted to 100 cc. Add a crystal of thymol and keep the bottle in the refrigerator.

Acetic acid, approximately 1.5 M; 42 cc. of 99.5 per cent acetic acid, diluted to 500 cc.

Ammonium hydroxide, 1: 1; 1 volume of ammonium hydroxide to 1 volume of distilled water.

Ammonium hydroxide, 1: 3; 1 volume of ammonium hydroxide to 3 volumes of distilled water.

Washing solution; 20 cc. of concentrated ammonium hydrox- ide in 980 cc. of a mixture of 1 volume of redistilled alcohol, 1 volume of redistilled ether, and 1 volume of distilled water.

Sulfuric acid, approximately 1 N; 27 cc. of concentrated sulfuric acid diluted to 1000 cc.

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446 Cn in Biological Material

Sulfuric acid, 1: 1; 1 volume of concentrated sulfuric acid to 1 volume of distilled water.

Nitric acid, concentrated. Nitric-perchloric acid mixture; 100 cc. of fuming nitric acid

(sp. gr. 1.49), 50 cc. of perchloric acid (sp. gr. 1.615), 100 cc. of distilled water.

Hydrochloric acid, approximately 20 per cent; 1 volume of concentrated hydrochloric acid to 1 volume of distilled water.

Hydrochloric acid, approximately 1 N; 100 cc. of concentrated hydrochloric acid diluted to 1000 cc. with distilled water.

Acid-washed charcoal.’ Standard potassium permanganate, 0.01 N (5). Standard sodium oxalate, 0.02 N; 0.134 gm. of sodium oxalate

dissolved and diluted to 100 cc.; Merck’s reagent, dried to con- stant weight and kept in a vacuum dessicator. It keeps about 1 month in the refrigerator.

Blank determinations must be made including all reagents in the quantities used in the actual determination. In the results reported here there was no detectable calcium in the reagents used.

All glassware, including reagent bottles, should be of Pyrex, ex- cept the burettes and pipettes. In this study Thomas pipettes were used.

Procedures

Blood Serum-To 1 volume of serum and 3 volumes of distilled water in an Erlenmeyer flask, is added, drop by drop with constant shaking, 1 volume of 20 per cent trichloroacetic acid. The mix- ture is allowed to stand for about + hour, transferred t.o a cen- trifuge tube, and centrifuged for 5 minutes. The supernatant liquid is poured off through a small ashless filter paper.

Into a scrupulously clean2 15 cc. conical centrifuge tube, with

1 We used Darco decolorizing carbon from the Darco Sales Corporation,

Sew York, but have not tested other charcoals. This carbon contained considerable calcium which was removed as follows: 10 gm. of the carbon

were boiled with about 150 cc. of 10 per cent HCl for about 10 minutes, poured on an ashless filter paper in a Buchner funnel, and washed with dis- tilled water until free of chlorides. It was then dried in an oven at about

100”. 2 The centrifuge tubes must be thoroughly clean, so that no liquid will

pling to the wall of the tube after decanting. It is advisable t.o boil the

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C. C. Wang

a proper point,3 5 cc. of the filtrate, 1 cc. of 20 per cent sodium acetate, 6 to 8 drops of 0.016 per cent brom-cresol green indicator, and 1 cc. of an approximately 0.1 M ammonium oxalate” are meas- ured. In adding the oxalate, care must bc taken that it drops directly into the solution and does not touch the wall of the tube, from which it will be difficult to remove by subsequent washings. The mixture is stirred with a fine glass rod and the pH is adjusted to match a phosphate buffer solution of pH 5 containing the same number of drops of the indicator in a similar volume, with 1: 1 ammonia at first and 1: 3 ammonia for the end-point.5 If the end-point is passed, the reaction is brought back to acid by means of dilute acetic acid, approximately 1.5 M. The rod is rinsed with water and the tube covered with a nursing bottle rubber cap. The mixture is permitted to stand for at least 1 hour, preferably overnight, and then centrifuged for 8 minutes at 2000 R.P.M. The supernatant solution is carefully decanted and while the tube is still inverted, it is placed in a rack on a few sheets of filter paper and allowed to drain for 5 minutes. (The supernatant liquid may be removed by suction with an immersion filter of a mesh that will hold back the precipitate and the immersion filter can be used afterwards as a stirring rod during titration.) The mouth of the tube is wiped with a strip of ashless filter paper and any drops that cling to the wall of the tube are also removed in this way. The precipitate is washed with approximately 3 cc. of

tubes first in a soap solution for a few minutes (Dreft or Orous are much better than ordinary soap), after which they are rinsed and heated at ap-

proximately 100” for a few minutes in a mixture of chromate and sulfuric acid.

3 The shape of the tube is important, because if the point is too narrow, a part of the fluid will cling to the bottom of the tube when being decanted.

If it is too wide, the precipitate will break. Since it is difficult to get the ideal shape and size of point, the simplest way is to test each tube with distilled water and see that all of the liquid can be removed by inverting the

tube slowly. * 0.1 M (NH&C201 deteriorates easily on standing at room temperature.

It should be kept in a refrigerator and preserved with a crystal of thymol. 5 In case the mixture is strongly acid, as in oxidizing food and feces, con-

centrated NH,OH should be used first, and if the reaction of the mixture is alkaline, it should be made acid with concentrated acetic acid before the addition of (SH4)nCr04.

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Ca in Eiological Material

2 per cent ammonium hydroxide in a mixture of equal parts of alcohol, ether, and distilled water from a wash bottle with a very fine jet, the stream being directed slightly above the point on the wall reached by the supernatant solution, while the tube is held at an angle of about 60” and is kept constantly rotating. The precip- itate is thoroughly broken with a fine Pyrex glass rod and mixed with the washing mixture. The glass rod is rinsed, removed, and the tube centrifuged for 5 minutes. The supernatant solution is again decanted and drained as above. This washing is repeated once and the tube is placed in an oven for about 1 hour at a tem- perature between 85-100’ in order to remove the last trace of the organic solvents. At the end of an hour, 2 cc. of approximately N sulfuric acid are delivered from a pipette into the tube, with care to wash the sides of the tube with the acid. The mixture is stirred with the fine glass rod and the tube is placed in a boiling water bath for 1 minute. The oxalic acid is immediately titrated with 0.01 N potassium permanganate,6 from a microburette graduated in 0.01 cc. and with a tip delivering not more than 0.015 cc. at a drop. If the oxalic acid requires more than 0.5 cc. of potassium permanganate, it is advisable to carry out the titration in a water bath at 70-75”. With the aid of a stirring rod it is possible to detect the end-point with not more than 0.008 cc. of the potas- sium permanganate. Thus the method is accurate from approxi- mately 0.0016 to 0.002 mg.

Urine-Fiske and Logan (6) have pointed out that calcium may be determined directly on urine only when it is quite free from material (other than calcium oxalate) which precipitates under the conditions used, and, that whenever such material is present ashing is either more convenient or necessary. Our ex- perience has been that, although in some cases it may be possible to determine calcium directly on fresh urine, it is not satisfactory, as high values frequently result. However, if the urine is treated with trichloroacetic acid, calcium values agreeing with those on urine ash are obtained. Even in apparently normal urine, with- out detectable traces of albumin, this treatment may remove interfering substances. One objection to using trichloroacetic

6 The author prefers using 0.01 N KMn04 made according to Halverson and Bergeim (5). When this solution is kept in a dark bottle, the concen- tration is not appreciably changed for at least a month and there is no necessity to restandardize in less than that time.

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C. C. Wang 449

acid filtrate for the permanganate titration method is the frequent presence of color in the filtrate which interferes with the adjust- ment of pH by brom-cresol green indicator. This difficulty is overcome by adding a small amount of acid-washed charcoal’ to the mixture of urine and trichloroacetic acid before it is filtered but after the volume has been made up. The filtrate from this mixture is usually clear and almost colorless.

Tests were made which proved that charcoal did not remove calcium from pure CaCL solution. In Table II, the results of parallel determinations on urine ash, trichloroacetic acid filtrate, and filtrate from trichloroacetic acid plus carbon are given. Prac- tically colorless urines were selected for this series. In the latter part of Table II are given a few representative parallel determina- tions on ashed urine and on trichloroacetic acid-carbon filtrate of urine so highly colored that determinations on the filtrate with- out carbon were impossible. It is evident that this treatment with trichloroacetic acid and carbon allows determinations of calcium in urine to within the accuracy of the titration technique. Incidentally, the use of carbon removes the objectionable urine odors.

Urine is treated with 20 per cent trichloroacetic acid in the same manner as is blood, except that the dilution should be varied ac- cording to the calcium content expected and carbon is added before filtering. For instance, in the urine of infants and that of patients suffering from certain diseases such as nephrosis, the total per 24 hour urinary calcium may be as low as 10 mg. On the other hand, the urinary calcium of adults and that of children suffering from progressive pseudohypertrophic muscular dystro- phy or in conditions of highly acid urine may be as high as 300 mg. per 24 hours. In the former case, 1 volume of 20 per cent trichloroacetic acid is added slowly to 4 volumes of urine in an Erlenmeyer flask. In the latter case, 1 volume of the acid is added to a mixture of 1 volume of urine and 3 volumes of water. For every 25 cc. of this trichloroacetic acid-urine mixture, about 0.4 gm. of acid-washed, dry charcoal is added. The mixture is shaken and allowed to stand for about 15 to 20 minutes with oc- casional shaking and then filtered through an ashless filter. The filtrate should be clear and almost colorless. Calcium is deter- mined on an aliquot 5 to 10 cc. portion of the filtrate as in the

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450 Ca in Biological Material

case of blood serum. Thus in the lowest output found, 10 mg. of Ca in 1000 cc. of urine per day, 10 cc. of the filtrate gave 0.08 mg.

TABLE II

Comparison oj Calcium Determinations on Ashed Urine with Those on Trichloroacetic Acid Filtrale with and without Charcoal

All precipitations stood overnight. The values are given in mg. per

100 cc

Faintly colored urines, 6 deter- minations on each

Highly colored urines, 2 deter- minations on each

LJrine NO.

1 2

3 4

5 6

7 8 9

LO 11 12-a*

12-b 13-a

13-b 14-a 14-b

15 16 17 18

19-ai 19-b

T i Med with 5 drops l?iltrate from

concentrated Trichloroacetic trichloroacetic HzS$h;a$ze;ec; UP acid filtrate acid filtrate +

charcoal

Range

11.4-11.1

Ll.6-ll.! Ll.l-Il.‘

LO.9-11.: 8.7- 9.t 8.6- 9.1

7.8- 8.: 7.8- 8.

_____ 4ver- we

RrtIl,BC?

11.6 11.5-ll.!

11.711.3-ll.! 11.3 10.5-11.

11.2 10.7-11.t 8.9 8.5- 8.1 8.8 8.5- 8.1

8.1 7.7- 8. 8.1 7.8- 8.

30.0 24.3 22.1 11.4

11.3 11.3

11.2 8.2

8.1 4.8 4.1 3.2 2.1

1.6 1.5

9 9 1

3 3 3

1 1 7

_____ 4ver- we

Range

__-___

11 .8 11.6-12.f

11.7 11.7-12.t 10.9 11 .o-11.:

10.9 11.2-11.: 8.7 8.5- 8.! 8.6 8.6- 9.f

7.9 7.7- 8. 7.8- 8.:

-

_ 4ver- age

11.8

11.8 11.2 11.2

8.7 8.8

8.0 7.9

30.4 24.4 22.1 11.4

11.2 11.3

11.2 8.2

7.9 4.9 4.0 3.1 2.0

1.6 1.6

* The a and b indicate separate days for the same subject. t Urine samples, from 2 successive days, from a child with nephrosis, on

an ordinary hospital diet. There were about 2 gm. of albumin per liter of urine.

of Ca, and in the adult urine of 300 mg. of Ca in 1500 cc. of urine per day, 5 cc. of filtrate gave 0.2 mg. of Ca. As shown in Table I, these amounts of calcium are accurately determinable.

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C. C. Wang 451

Food and Feces-About 2 gm. of dry, finely ground food or 0.5 gm. of feces is placed in a 250 cc. Kjeldahl flask (or 800 cc. flask if perchloric acid is employed). It is oxidized with either sulfuric and nitric acids or by a nitric-perchloric acid mixture ac- cording to the method of Lematte, Boinot, and Kahane (7).

Sulfuric Acid Method-10 cc. of 1: 1 sulfuric acid are placed in a flask containing the dried material and the flask is heated over a water bath until the mixture is thoroughly charred. Nitric acid, 8 to 10 drops at a time, is added from time to time and the flask shaken and heated over a microburner until the mixture is thoroughly oxidized and becomes colorless on further heating. The solution is cooled and rinsed into a 500 cc. volumetric flask and made up to the mark with distilled water. In order to re- move the excess of sulfate, an aliquot part, usually 5 cc., of the solution is pipetted into a 30 cc. Pyrex beaker, evaporated down to dryness on a water bath, and placed in a muffle furnace at 600' for 15 to 20 minutes. Onto the residue 1 cc. of 20 per cent hydrochloric acid is measured and evaporated just to dryness on a sand bath on a electric plate, care being taken to avoid spat- tering. About 10 minutes are usually required to bring the solu- tion just to dryness. This procedure is repeated four times, the beaker being cooled each time before the addition of hydrochloric acid. The final residue is dissolved in 2 cc. of N hydrochloric acid and is quantitatively transferred into a 15 cc. conical centrifuge tube. The procedure for the precipitation of calcium is then car- ried out as described above for serum.

Nitric-Perchloric Acid Method-To the flask containing the dry material are added about 40 cc., or 20 cc. in the case of feces, of the nitric-perchloric acid mixture and the flask is heated over a microburner with constant shaking in order to avoid explosion. When white fumes begin to appear and the liquid starts to darken, more acid mixture, 10 to 20 cc., is added. The digestion is continued until the mixture becomes colorless or only slightly colored. The flask is cooled and the mixture is quantitatively transferred into a 500 cc. volumetric flask. Calcium is deter- mined on 5 cc. portions of the solution. Although it requires considerable care to prevent explosion, this method saves the step of removing the excess of sulfate ion.

Interference of Sulfate-One of the difficulties encountered in

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452 Ca in Biological Material

determining calcium in urine or in the ash of the digestion by means of sulfuric and nitric acids is the interference of the sulfate ion in the precipitation of calcium oxalate noted by Fiske. In order to find the proportion of sulfate to calcium at which the former would not interfere appreciably with the precipitation under the conditions of oxalate concentration suitable for this new washing technique, experiments were made in which various amounts of sulfuric acid were added to serum filtrates of known calcium value. As shown in Table III, the interference decreases

TABLE III

Effect of Sulfate Ion on Calcium Oxalate Precipitation

Calcium and HzSOa were added to tubes containing 5 cc. of trichloro-

acetic acid filtrate of dog serum equivalent to 1 cc. of serum, containing 0.098 mg. of calcium oxalate added; pH adjusted to 5, and allowed to stand

overnight.

Ca added Total Ca present

w. mg. 0.062 0.160 0.124 0.222

0.186 0.284

0.062 0.160 0.124 0.222

0.186 0.284 0.062 0.160 0.124 0.222

0.186 0.284 0.124 0.222 0.186 0.284

mg.

432.5 2703 432.5 1948 432.5 1523 288.3 1802 288.3 1299

288.3 1015 216.3 1352 216.3 974

216.3 761 144.2 649 144.2 506

-

w?.

0.080 0.148 0.230

0.131 0.201 0.272

0.147 0.214

0.275 0.218 0.281

per cent 50.0 33.3 19.0 18.1

9.5 4.2

8.1 3.6

3.2 1.8 1.1

3ulfate added

-7 - --

Deviation from total 104 : Cs ratio

with the ratio of sulfate to calcium, and when the ratio is approxi- mately 500: 1, the interference becomes negligible. When inter- ference occurs, multiple determinations disagree among them- selves and failure to get good checks should raise suspicions as to completion of precipitation. In our experience, whenever sulfuric acid is used in oxidation, unless the ratio can be dennitely established by calculation, it is wise to remove the excess of sulfate by means of heat and to convert the insoluble metaphosphates to soluble orthophosphates first, otherwise the results may be considerably lower than the actual values. Although a ratio of

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C. C. Wang

50O:l seems rather large, it does not take much sulfuric acid to reach this point. For instance, in Fiske’s alkalimetric calcium method (6), when he oxidizes a urine specimen containing from 0.25 to 0.75 mg. of calcium with 1 cc. of 10 N sulfuric acid, not including the sulfate already present in the specimen, the ratio lies between 1920 and 640 to 1.

This high sulfate-calcium ratio does not interfere with accurate results in Fiske’s method, but it is apparent that it does under the conditions of lower oxalate concentration used with the present method. Since the sensitivity to sulfate interference is increased under these conditions, it is possible that the interfering action of other substances, as P and Mg discussed by Fiske, might likewise be intensified. The result on artificial tissue salt mixture, given in Table I, shows that salt relations in tissue ex- tract or ash do not interfere and the results with urines given in Table II indicate that there is no interference in ordinary human urine. We have not yet tested it against Fiske’s double precipi- tation method for urines containing unusually high phosphorus and magnesium.

SUMMARY

1. A new washing solution for calcium oxalate precipitation of 2 per cent ammonia in equal parts of alcohol, ether, and water prevents flotation and permits washing of the precipitate wit.hout appreciable loss of calcium.

2. The treatment of urine with trichloroacetic acid and carbon allows direct calcium determination on urine.

BIBLIOGRAPHY

1. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 47, 475 (1921); 48, 223 (1921).

2. Clark, E. P., and Collip, J. B., J. Biol. Chem., 63,461 (1925). 3. Van Slyke, D. D., and Sendroy, J., Jr., J. Biol. Chem., 84,217 (1929). 4. Clark, W. M., The determination of hydrogen ions, Baltimore, 3rd edi-

tion, 61, 94, 126 (1928). 5. Halverson, J. O., and Bergeim, O., J. Ind. and Eng. Chem., 10,119 (1918). 6. Fiske, C. H., and Logan, M. A., J. Biol. Chem., 93,211 (1931).

7. Lematte, L., Boinot, G., and Kahane, E., J. pharm. et chim., 6, 325, 361 (1927).

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Chi Che WangBIOLOGICAL MATERIAL

FOR CALCIUM DETERMINATION IN IMPROVEMENTS IN THE METHODS

1935, 111:443-453.J. Biol. Chem. 

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