THE DIRECT QUANTITATIVE DETERMINATION OF

SODIUM, POTASSIUM, CALCIUM, AND MAGNE-

SIUM IN SMALL AMOUNTS OF BLOOD.
BY BENJAMIN KRAMER AND FREDERICK F. TISDALL.
(From the Department of Pediatrics, The Johns Hopkins University,
Baltimore.)

(Received for publication, June 8, 1921.)

The concentration of sodium, potassium, calcium, and magnes-

ium in the blood of animals has been determined by Bunge (I),
Abderhalden (Z), and more recently by Greenwald (3). These

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

investigators used from 25 to 100 cc. of blood for their deter-
minations. Such quantities of blood cannot conveniently be
used in studies with patients, particularly children. We have
therefore devised a method by means of which the concentration
of all these elements may be quantitatively determined on 7 cc.
of blood.
Principle of the Method.
Deproteinization is carried out by means of the trichloroacetic
acid method recommended by Greenwald (4). The determina-
tions of the individual elements, except that of magnesium,
are then made directly on separate aliquots of the deproteinized
fluid by modifications of methods previously decribed by us for
serum (5, 6, 7). The inorganic or acid-soluble phosphorus may
also be determined by any of the well known micro methods
(8) on a portion of the supernatant fluid corresponding to 0.5
or 1.00 cc. of blood.
Methods,
Collection of Material and Deproteinization.-25 cc. of distilled
water are placed in a 50 cc. volumetric flask which is then weighed.
From 7 to Sg cc. of blood are obtained by means of a 10 cc. grad-
uated syringe and slowly added to the water in the flask. The
flask should be continuously rotated during this procedure which
223
224 Inorganic Elements of Blood

completely hemolyzes the blood. The flask and contents are

again weighed and the exact amount of blood added thereby
determined. 1 or 2 drops of octyl alcohol are added followed
by 12 to 13 cc. of 12 per cent trichloroacetic acid which are added
slowly while the flask is rotated. The contents are thoroughly
mixed and allowed to stand 10 minutes. Water is added to 50 cc.,
the contents are again mixed, transferred to a large centrifuged tube
and centrifuged for 5 to 10 minutes at about 1,000 revolutions per
minute. The supernatant fluid is poured off and an aliquot (gener-
ally 35 cc.) is placed in a beaker and evaporated. The presence of

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

of a few particles of the precipitate which sometimes float on
the surface of the supernatant fluid does not interfere with the
subsequent determinations. If the particles are very numerous,
the fluid may be allowed to stand in the ice chest for a few hours.
They will then have settled to the bottom. The supernatant
fluid may be kept at this stage for at least 2 weeks before com-
pleting the determination. After the aliquot has been evapo-
rated t’o dryness the residue is dissolved in 0.1 N HCI and is
transferred to a volumetlric flask and the volume made up to 10
cc. This fluid has the appearance of serum. Should it be cloudy
it may be centrifuged for a few minutes when a clear, straw-
colored supernatant liquid will be . obtained. The sodium,
potassium, and calcium determinations are done directly on this
material while the magnesium determination is done on the first
supernatant fluid obtained from the calcium determination.

Sodium Method.
4 cc. of the material prepared as outlined above are placed
in a platinum dish and evaporated to about 2 cc. A drop of
phenolsulfonephthalein is added and the contents are made just
alkaline with 10 per cent KOH (generally about 10 to 12 drops
will suffice). 30 cc. of the potassium pyroantimonate reagent
are added followed by 3 cc. of 95 per cent alcohol. The alcohol
should be added drop by drop and the specimen stirred with a
rubber-tipped rod. After standing 30 minutes the precipitate
is transferred to a weighed Gooch crucible and washed with 5
to10 cc. of 30 per cent alcohol. The crucible is dried at 110”
C. for 1 hour,l cooled in a desiccator for 30 minutes, and weighed.
1 The temperature should be gradually raised to 11O’C.
 B. Kramer and F. F. Tisdall 225

The weight of the precipitate divided by 11.08 equals the number

of mg. of sodium present in the sample.
The method of preparation of the potassium pyroantimonate
reagent has been fully described in a former paper on the deter-
mination of sodium in serum (5). The details of the method
of preparation of the Gooch crucibles and the precautions to be
observed during the addition of the alcohol and the filtration,
and also the care of the platinum are fully outlined in the same
paper.
For the determination of sodium in solutions of blood ash we

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

used the same procedure as described for the determination of
this element in solutions of the ash of urine and stools (9).

Potassium Method.
0.2 cc. of the material prepared as previously outlined is placed
in a graduated centrifuge tube. 0.5 cc. of water is added followed
by 0.5 cc. of a solution of sodium nitrite prepared by dissolving
15 gm. of potassium-free sodium nitrite (Merck) in 30 cc. of water.
The contents of the tube are thoroughly mixed and allowed to
stand for 5 minutes.2 Water is added to 4 cc. and the contents are
again mixed. 2 cc. of the sodium cobalti-nitrite reagent are
added drop by drop. The contents of the tube are mixed and
allowed to stand for a half hour, then centrifuged for 7 minutes
at about 1,300 revolutions per minute. The precipitate will
then be found at the bottom of the tube. All but 0.2 to 0.3 cc.
of the supernatant fluid is removed. This is accomplished by
means of the following apparatus. Through one opening of a
two-holed cork is inserted a glass tube by means of which a
positive pressure can be made in the centrifuge tube. Through
the other hole is a tube which reaches to about 3 or 4 mm. above
the precipitate. The lower end of this tube is drawn out to a
2 If the sodium nitrite is not added, it will be found that the precipi-
tate obtained on the addition of the cobalti-nitrite reagent will float on
the surface of the fluid and adhere t.o the sides of the tubes. The precip-
itate will also adhere to the sides unless the tubes have been previously
cleaned with the use of a brush, washed out with a strong cleaning fluid
(commercial H&304 and dichromate) and then thoroughly rinsed with
distilled water. Low results will be obtained unless these procedures
are carried out.
 Inorganic Elements of Blood

bore of about 1 mm. and curved so t,hat the opening is directed

upward. By fitting the cork to the centrifuge tube and blowing
through t’he first opening the supernatant fluid can be readily re-
moved without disturbing the precipitate. 5 cc. of water are
allowed to run down the side of the tube which is then gently
agitated so that the added water is mixed thoroughly with the
residual reagent. Care should be taken that the precipitate itself
is disturbed as little as possible. This may be accomplished by
holding the tube vertically and gently hitting the lower end
with a circular motion. The brown fluid may be seen to rise

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

and mix with the supernatant fluid. The tube is then centri-
fuged for 5 minutes. The procedure is repeated three times
so that the precipitate is washed four times in all. The superna-
tant fluid from the last washing should be perfectly clear. After
the removal of the fluid from the final washing the precipitate
is ready to be titrated.
Titration.-An excess of 0.02 N potassium permanganate is
added (1.6 to 2 cc. are sufficient for normal blood), followed by
1 cc. of approximately 4 N sulfuric acid. The precipitate is
then t,horoughly mixed with the fluid by means of a glass rod.
The sample is heated in the boiling water bath for 45 to 60 seconds
at the end of which time the solution should be clear and still
pink. If all the precipitate is not oxidized, the contents will
be cloudy and the intensity of the color will be seen to diminish.
Heating should then be continued until the solution is clear
but still pink. When the heating is continued too long, the
contents again become cloudy and have a brownish color. If
this is allowed to happen, the sample must be discarded as high
results will be obtained. An amount of 0.01 N sodium oxalate
sufficient to decolorize the solution completely (generally 2 cc.)
is promptly added. The excess of oxalate is then determined
by titrating to a definite pink color with 0.02 N potassium per-
manganate delivered from a micro-burette graduated in 0.02 cc.
The details for the calculation of the amount of potassium
present in the sample and also the methods for the preparation of
the reagents are given in a former paper (9).
For the determination of potassium in solutions of blood ash
we placed a quantity of fluid equal to 0.1 or 0.2 cc. of blood in
a graduated centrifuge tube, added water to 2 cc., and then
 B. Kramer and F. F. Tisdall 227

slowly added 1 VC. of the cobalti-nit,rite reagent. The subsequent

steps were the same as described for the determination of t,his
element in serum (5).
Calcium Method.

4 cc. of t,he material prepared as outlined above are placed

in a graduated centrifuge tube previously cleaned wit’h commer-
cial HzS04 and potassium dichromate. 1 cc. of saturated ammo-
nium oxalate is added, followed by 2 cc. of a filtered sat,urated
solution of sodium acetate. The contents are mixed and allowed

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

to stand for 1 hour. The volume is made up to 8 cc. wit,11 dis-
tilled water, mixed, and then cent)rifuged for 15 minutes at about
1,300 revolutions per minute. This throws all the calcium
oxalate precipit,ate t,o the bottom of the tube. All but 0.3 cc.
of the supernat,ant fluid is removed by means of the apparatus
described under t,he potassium method. The remaining fluid
and the precipitate are mixed by t’apping the tube. Enough
2 per cent ammonia (2 cc. of concentrated ammonia diluted to
100 cc.) is then added to bring the volume to 4 cc., care being
taken to wash t,he sides of the centrifuge tube free from adherent
oxalic acid. The tube is then centrifuged for 5 minutes. This
procedure is repeated t,wice, thus making three washings in all.
After the third washing the supernatant fluid is removed, the
tube is shaken to suspend the precipitate, 2 cc. of approximately
K sulfuric acid are added and t,he tube is warmed in the boiling
water bath for a few minutes and titrated with 0.01 N potassium
permanganate until a definite pink color persists for at least 1
minute when viewed under a good light against a white back-
ground. The strength of the permanganate solution is determined
by titrating against a 0.01 N sodium oxalate (Sorensen).
The details for the calculation of the amount of calcium present
in the sample and also the methods for the preparation of the
reagents are given in a former paper (9).
For t’he determination of calcium on 0.1 x HCl solutions of
blood ash the procedure is identical with that described above.
The blood ash solution was made up so that 1 cc. corresponded
to 1 cc. of blood. It was allowed to stand for 2 or 3 weeksto allow
the sediment to set.tle. 2 cc. of this solution were used for each
determination.
228 Inorganic Elements of Blood

The Magnesium Method.

5 cc. of the supernatant fluid from the calcium determination

are measured into a 30 cc. beaker, 1 cc. of (NH&HPO, solution
is added and then 2 cc. of concentrated ammonia. The next
day the sample is filtered through a well packed Gooch crucible,
washed ten times with 5 cc. of 10 parts of concentrated ammonia
to 90 parts of water, then twice with 95 per cent alcohol made
alkaline wit.h ammonia. The crucible is returned to the beaker
and dried for a few minutes at 80” C. in the oven.

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

10 cc. of 0.01 N HCl are added to the crucible and after a few
hours the entire material is transferred to a test-tube, centrifuged,
and 5 cc. of the supernatant fluid are measured into a flat bot-
tomed calorimeter tube graduated for 10 cc., which contains
2 cc. of the iron thiocyanate solution. The volume is then made
up to 10 cc. with 0.01 N HCl, a rubber stopper inserted, and the
fluid mixed. A series of standards is prepared by adding varying
amounts of a known NH4MgP04 solution in 0.01 N HCl, to 2 cc.
samples of thiocyanate solution and bringing the volume up to
10 cc. as in the unknown samples. The color is compared by
looking through the entire length of the liquid column against
a white background.
Calculation.-The calculation is the same as in the original
method: Reading (cc. of standard solution) X0 .Ol ~2 x8/5 X
100
= mg. of magnesium in 100
cc. blood used in Ca determination
cc. of blood.

Preparations of Reagents.

1. Ammonium Magnesium Phosphate Standard.-This solu-

tion is made by dissolving 0.102 gm. of air-dried magnesium
ammonium phosphate (MgNH,P04.6H20) in 100 cc. of 0.1 K
hydrochloric acid and diluting to 1 liter with water. Of this
solution 1 cc. is equivalent to 0.01 mg. of magnesium. Mag-
nesium ammonium phosphate loses water of crystallization
when heated and must therefore be dried at room temperature.
Commercial preparations of the salt are generally unreliable; it
should be prepared by precipitation of pure solutions (10).
 B. Kramer and F. F. Tisdall 229

2. Ammonium, Phosphate Solution.-Ammonium phosphate

solution is made as follows: 25 gm. of (NH&HPOI are dissolved
in 250 cc. of HzO. 25 cc. of concentrated ammonia are added
and the mixture is allowed to stand over night. The following
day it is filtered, the filtrate is boiled to remove the excess of
ammonia, cooled, and made up to 250 cc. This solution is
diluted five times with water.
3. The Ferric Thiocyanate Xolution.-This solution is made
from two solutions which are mixed an hour before use. Solu-
tion A is 0.3 per cent ammonium thiocyanate. Solution B is

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

0.3 per cent ferric chloride, made up from the salt with its con-
tained water of crystallization, adding a few drops of acid,
if necessary, to clear the solution. 5 cc. portions of Solutions
A and B are mixed and the whole is diluted to 40 cc. wit’h water.
4. 10 Per Cent Ammonia.-100 cc. of concentrated ammonia
are diluted to 1 liter.
Protocols.

We have previously shown (5) that sodium may be precipi-

tated quantitatively, as sodium pyroantimonate, from solutions
of blood salts. Since the composition of the supernatant fluid
after deproteinizing blood with trichloroacetic acid is comparable,
except for the small amount of residual protein and the non-
protein nitrogenous constituents, to some of the solutions of
blood salts which we have analyzed, we have considered it
unnecessary to repeat this demonstration here.
Table I shows that the sodium determinations when performed
on the deproteinized solutions yield results practically identical
with those obtained on a solution of the whole blood ash. The
absolute values vary from 170 to 225 mg. of sodium per 100 cc.
of blood.
Table II. The concentration of potassium seems to vary
considerably in the blood of different animals. Bunge found
213 mg. of potassium in 100 cc. of pig’s blood, 227 mg. in that of
the horse, but only 34 mg. in the same volume of cow’s blood and
20 mg. per 100 cc. of dog’s blood. The lowest figure which
Abderhalden reports is 21 mg. of potassium per 100 cc. of dog’s
blood while the highest figure is 227 mg. per 100 cc. of horse’s
blood. We have found that the potassium content of human
230 Inorganic Elements of Blood

blood varies from 153 t’o 202 mg. of potassium per 100 cc. It
varies with the percentage of corpuscles. It might be mentioned
that the cobalti-nitrite reagent gives no precipitate when added
to ferric chloride or trichloroacetic acid. We have shown else-
where t,hat none of the constituents of serum except potassium
TABLE I.

Sodium Determinations on Blood.

;a per 100 cc of blood
Samplt-. Plasma. deproteinirrd

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

per cent “W
65 216
55 173
65 XI07
58 186
56 193
60 200
l-
Average. I 60
195 196

TABLE II.

Potassium Determinations on Blood.

Determinations on blood treated with

Determinations on ashed b!ood. trichloroacetic acid.

Ii per loo cc. Ii per 100 cc

Sample. Ssmple. Plasma.
of blood. of blqod.
-I-
m*. rv

1 61 172 7 61 180
2 60 187 8 6”s Ii.5
3 57 188 9 ~ 57 20’7
4 1 68 153 10 5!? 193
5 58 186 11 65 164
6 5f 200 12 65 169
13 56 201

Average.. .j 60 181 61 183

yields demonstrable amounts of insoluble nitrites with this reagent

(6).
Table III. The concentrat,ion of calcium in serum or plasma
is singularly constant (7). On the other hand the concentration
of this element in blood varies inversely as the corpuscular con-
 B. Kramer and F. F. Tisdall 231
tent. The results which we obtained varied from 5.3 to 6.8 mg.
of calcium per 100 cc. of blood. The individuals from whom the
samples were obtained were all normal adults. We have found
that the addition of ferric chloride to a solution of blood salts
TABLE III.

Calcium Determinations on Blood.

Determinations on ashed blood Determinations on blood treated with

trichloroacetic acid.

Sample. Plasma. Ca per 100 cc. Sample Plasma

I’l-
“a per 100 cc.

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

of blood. of blood.

per cent ,,LQ. per cent mg.

1 58 5.3 8 6.3
2 57 5.3 9 5.3
3 72 6.7 10 6.1
4 59 6.2 11 5.3
5 58 5.3 12 5.7
6 65 5.9 13 G.4
7 57 5.5

Average.. 5.i 5 .8

TABLE IV.

Magnesium Determinations on Blood.

Determinations on blood treated with
Determinations on ashed blood. trichloroacetic acid.

Mg. per 100 cc. of

Sample. Sample. blood.

mQ rnQ.
1 2.8 5 2.6
2 2.8 6 4.0
3 3.8 7 3.8
4 2.3 8 3.8

Average 2.9 3.5

does not interfere with the quantitative determination of calcium.

The results obtained on the deproteinized material and on the
solutions of blood ash are practically identical.
Table IV. The concentration of magnesium in the blood of
various animals has been found by Bunge and Abderhalden
232 Inorganic Elements of Blood

to be fairly constant, varying only from 2 to 4 mg. per 100 cc. of

blood (2). We have found that the concentration of this element
in the blood of the adult male varies from 2.3 to4.0 mg. per 100 cc.

CONCLUSIONS.

1. A method has been described by means of which sodium,

potassium, calcium, and magnesium may be quantitatively deter-
mined on only 7 cc. of blood.
2. The basis of this method is deproteinization by means
of trichloroacetic acid. The quantitative determination of each

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

of these elements is then made on aliquots of the supernatant
fluid by modifications of procedures recently described for serum.
3. The results obtained by these methods on deproteinized blood
agree well with those obtained on solutions of blood ash.
4. We have found the concentratjion of these elements in 100
cc. of human blood to be as follows: sodium, 170 to 225 mg.;
potassium, 153 to 201 mg.; calcium, 5.3 to 6.8 mg.; and mag-
nesium, 2.3 to 4 mg.
5. The concentration of these elements in normal blood varies
more than in normal serum. This is due to the variations in
the corpuscular content of the blood.
BIBLIOGRAPHY.

1. Bunge, G., 2. Biol., 1876, xii, 191.

2. Abderhalden, E., 2. physiol. Chem., 1897, xxiii, 521; 1898, xxv, 65.
3. Greenwald, I., J. Pharmacol. and Exp. Therap., 1918, xi, 281. Green-
wald, I., and Gross, J., Proc. Sot. Exp. Biol. and Med., 1919-20,
xvii, 50.
4. Greenwald, I., J. Biol. Chem., 1915, xxi, 61.
5. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlvi, 467.
6. Kramer, B., andTisdal1, F. F., J. Biol. Chem., 1921, xlvi, 339. How-
land, J., and Marriott, W. NICK., Quart. J. Med., 1917-18, xi, 289.
Kramer, B., and Howland, J., J. Biol. Chem., 1920, xliii, 35.
7. Kramer, B., and Tisdall, F. F., Bull. Johns Hopkins Hosp., 1921,
xxxii, 44.
8. Greenwald, I., J. Biol. Chem., 1915, xxi, 29. Marriott, W. McK., and
Howland, J., J. Biol. Chem., 1917, xxxii, 233. Bloor, W. R., J. Biol.
Chem., 1918, xxxvi, 49. Feigl, J., Biochem. Z., 1917, lxxxi, 380.
Iversen, P., Biochem. Z., 1920, cix, 211. Kleiman, H., Bio:hem. Z.,
1919 xcix, 19.
9. Tisdall. F. F., and Kramer, B., J. Biol. Chem., 1921, xlviii, 1.
10. Jones, W., J. Biol. Chem., 1916, xxv, 87.
 THE DIRECT QUANTITATIVE
DETERMINATION OF SODIUM,
POTASSIUM, CALCIUM, AND
MAGNESIUM IN SMALL AMOUNTS OF
BLOOD
Benjamin Kramer and Frederick F. Tisdall
J. Biol. Chem. 1921, 48:223-232.

Downloaded from http://www.jbc.org/ by guest on February 3, 2019

Access the most updated version of this article at
http://www.jbc.org/content/48/1/223.citation

Alerts:
• When this article is cited
• When a correction for this article is posted

Click here to choose from all of JBC's e-mail

alerts

This article cites 0 references, 0 of which can be

accessed free at
http://www.jbc.org/content/48/1/223.citation.full.ht
ml#ref-list-1