TITRATION OF THE CALCIUM-BINDING CAPACITY OF URINE1

By REIDH. LEONARD,Ph.D., and ARTHUR

J. BUTT, M.D.
Pensacola. Florida

IN renal calculous disease some derangement of the ordinary urine composition is thought to
exist. There are relatively few chemical methods which provide specific evidence for evaluation
of stone-producing conditions. For example, elevated urinary cystine or uric acid are good
indicators of the type of stone formed. However, most stones are calcium salts and no clear-cut
predictions can be made from the urinary calcium and phosphorus contents. Urinary oxalic
acid is seldom determined as an indicator of stone type.
TABLEI
Calcium Content of Calculi
Calcium Content Weight
per Cent.
Over 35
30-1 to 35
25.1 to 30
20.1 to 25
15.1 to 20
10.1 to 15
5.1 to 10
0.1to 5
0.0 .

.
.
.
.
.
.
.

.
.

Number.

3
32
174
111
31
11
4
20
12
398

Per Cent.
I

I
8
43
28
8
3
1
5

3
100

The analyses of 398 stones by chemical methods show that 78 per cent. are composed of
calcium oxalate or calcium phosphate in various proportions and that 91 per cent. contain over
10 per cent. calcium. This classification of stones by their quantitative calcium content is shown
in Table I. In addition, chemical analysis indicates a somewhat random ratio of calcium to
phosphorus which shows that the pure compounds ordinarily considered as stone components
seldom exist as single components but rather as mixtures of components. The random ratio
of calcium to phosphorus is more in harmony, from the chemical viewpoint, with the known
behaviour of calcium phosphate precipitates than is the single compound structure.
These suggestions from chemical analysis indicate that the formation of stones results from
some disorder in the ordinary behaviour of calcium in the urine ; that the anions of the stone
salts, oxalate, phosphate, and carbonate are incidental to the ability of the calcium to become
attached to them. Urine differs from a mixture of simple salts in solution in that significant
quantities of many organic materials are present. The incongruous existence of calcium,
phosphate, and oxalate in common solution in urine can be explained by assumption of the
existence of calcium compounds which are soluble and yet do not ionise appreciably. These
compounds could be calcium salts which ionise only slightly in the presence of the ionic
Abridged from addresses presented at the Institute of Urology (University of London), University of Leeds,
and Queens University, Belfast.
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T I T R A T I O N OF THE C A L C I U M - B I N D I N G

C A P A C I T Y O F URINE

28 1

constituents of urine or they could be calcium compounds of the chelate form, heterocycles
with polyvalent attachment of calcium. The presence of non-ionising calcium compounds in
urine has been demonstrated by ion exchange and by dialysis techniques.
There are several methods of assaying the calcium binding or calcium chelating capacity
of a solution. Vermeulen et al. (1956) describe ion exchange and dialysis; methods of
determining the pCa are reviewed by Raaflaub (1956), and Martel and Calvin (1952) describe
general methods of measuring chelates. The application of these various methods to urine
is limited, since many specimens are turbid and accuracy of the method may be reduced by
removing part of the material in the sample by clarification steps. Any method, in addition,
should measure any calcium-binding effect at the pH of the natural urine and not at abnormal
ranges, and it should not be limited to any particular molecular size. One possible method
is by the addition of a neutral calcium salt to the urine and observation of the pH shift. Some
phases of this process have been examined in urine with evidence that such a calcium-binding
system exists and is influential in calculus formation.
TABLEI1
Change in Urine pH upon Addition of Calcium Chloride
Initial pH of Specimen.

Decrease in pH after
addition of CaCl,.

Zalcium-binding Capac it
at pH 6.5 mEq./litres.

8.52
5.68
6.08
5 40
5 60
6.60
5-32
6 .O
8.40
5.60
7.50
7.02

1.16
0.35
0.71
0.27
0.35
0.68

10.5
9.2
13.6
12.7
12.6
9.0
16.5
10.7
0.9
15.0
13.5
20.2

0.40
0.44
0.80
0.39
1.38
1.20

Change in pH with added Calcium Salt.-Addition of neutral calcium chloride solution to

urine causes a decrease in pH. This can be accounted for by the presence of a substance (CBC)
which is capable of holding calcium ions in preference to hydrogen ions.
Eq. 1.
nCa++ CBC + CBC
2nH+
(H form) . (Ca form)

Decrease in p H means that only the hydrogen ion is displaced by the added calcium, that
the calcium present in the urine is in equilibrium with these substances, and that all such materials
are not ordinarily saturated with calcium. Some results on different individuals are shown in
Table 11. These values were obtained by adding 5 ml. of neutral 10 per cent. calcium chloride
to one-hundredth aliquot of a twenty-four-hour urine specimen. The decrease in pH after mixing
was 0.3 to 1.4 units and has shown no relationship to the calcium-binding capacity.
Quantitative Titration.-An
accurate aliquot of a twenty-four-hour urine specimen of
approximately one-hundredth volume is placed in a beaker containing a magnetic stirrer and
the electrodes of a p H meter (Beckman Model G ) . Then with stirring either a measured amount
of 2N HCI or one or two drops of concentrated HCI are added to lower the pH to about 3, and
3c

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the acidified sample is titrated to pH 9 with 0.25 N KOH, the latter being added in 0.5 ml.
increments. ThepH is recorded after each increment. The sample is then discarded and a fresh
aliquot supplied. To this is added 5 ml. of neutral 10 per cent. CaCl, and the same amount of
HCI to lower the pH. A second titration is made and recorded.
The values from the two titrations are then plotted against alkali to make a pair of curves,
as shown in A and B of illustration. Except for a slight horizontal displacement the two curves
are identical in form below pH 5 or 5.5. This identity in form in this pH region is characteristic
of every urine specimen thus far encountered. A second characteristic is the deviation of curve B
which is found with calcium chloride present in the titration. The deviation always starts at

"

'

"

ML.

'
'
KOH

"

'

Titration of a pooled urine specimen. Curve A obtained by titration of

urine ; curve B obtained with ,calcium chloride present. Tie-lines show
quantitative measures.

about pH 5 or 5.5 and reaches a maximum at about pH 6.5. In the illustration the deviation of
curve B away from curve A at pH 7 is sufficient evidence that additional alkali must be added,
as shown by the line Y connecting A and B at pH 7. By measuring lines x, N, Y, and z the
amount of standardised alkali is obtained which represents the calcium-binding capacity of the
urine at pH 6, 6.5, 7, and 8 respectively. The final units are either milliequivalents per twentyfour hours or per litre. At higher pH values the two curves tend to close, although seldom
becoming identical below pH 10.
In practice the two curves are plotted on the same paper in reasonable proximity to each
other and with the pH scale invariable. Then by means of dividers the millilitres of alkali at
pH 5.5 and at yH 6.5 are measured. The alkali consumed at pH 5.5 is subtracted from that
for pH 6-5 and the difference computed as milliequivalents of calcium-binding capacity. With
care the results are reliable to about 2 mEq./litre.
In order to arrive at some reasonable cause for the paired curves similar titrations were made
with several pure compounds. Curve pairs with the general shape of urine were obtained with
monopotassium phosphate and with sodium phytate. However, the maximum deviation between
the curves of orthophosphate falls at pH 5.5 and for phytate at pH 5 as compared to urine at
pH 6.5. Consequently, if the measurements of alkali consumption are zeroed at pH 5.5 the
effect of orthophosphate is eliminated in the calculations.
Either the phosphate ion plays a minor part in the behaviour of the urine or else it is
modified by some other material in the urine. The titration of a mixture of monopotassium
phosphate and glycine yielded a pair of curves more nearly resembling those obtained in urine.

TITRATION OF

THE

CALCIUM-BINDING

CAPACITY

283

OF U R I N E

It is possible that a very effective calcium solubilising system is produced by amino compounds
and phosphate.
The selection of pH 5.5 as a zero point and pH 6.5 as the best measuring pH was made
empirically after examining over 100 specimens at pH values from 5 to 8.
RESULTS
Determination of the calcium-binding capacity has been made in over forty patients at
intervals of several months. Table 111 gives results on two patients. Values of calcium-binding
capacity are given in three different terms : milliequivalent per twenty-four hours, milliequivalent
per gram of urinary phosphorus, and milliequivalent per milliequivalent of urinary calcium.
Included in the table are results obtained when the patients received, variously, acetylsalicylic
acid, salicylamide, hyaluronidase, sodium phytate, and monosodium glutamate.
TABLEI11
Calcium-binding Capacity of Two Patients
Calcium-binding Capacity 5.5 to 6.5.
Patient .

TD .

AT

Date.

27/2/56
28/2/56
29/2/56
5/3/56
27/8/56
28/8/56
29/8/56
31/8/56
8/1/57
9/l 157
10/1/57
27/3/57
10/5/57
8/10/56
9110156
11/10/56
17/10/56
18/10/56
29/11/56
3/2/57
10/4/57

Treatment.

...
H
A
H+A

...

A
SP
H+A

...

H
MSG
MSG
MSG

...
H

SP
H
H+SA

...

M SG

...

H =Hyaluronidase.
A = Acetylsalicylic acid.
SP=Sodium phytate.

Volume.

1,540
1,240
2,580
3,480
1,730
2,685
2,100
2,050
2,240
2,210
2,280
3,280
2,750
2,130
2,180
2,050
2,170
2,910
1,510
1,580
1,060

nEq.124 hours.

mEq./g. P.

mEq./mEq. Ca.

15.4
18.2
17.2
40.2
20.9
19.7
30.3
21.9
25.7
29.5
31.6
29.4
294
1.9
0 .o
5.9
7.3
7.9
19.5

...
...

...
...

21 . I
19.5
46.0
43 .O
22.5
26.1
28.7

3.5
1.6
3.6
2 .o
4 .O
5.4

10.0

11.1

...
...

...

...

...

5.1

...

...
...

76

0.3

22.7
29.2
28.2
48.7

2.6
2.2
1.7
4.5

...

3 .O

...

...

...

...

MSG =Monosodium glutamate.

SA= Salicylamide.

Case T. D. showed a gradual increase in calcium-binding capacity over a years time which
may indicate a favourable effect of drugs upon this capacity. This increase in binding capacity
followed an increase in total urine volume. Sodium phytate and hyaluronidase plus
acetylsalicylic acid induced a significant increase in calcium-binding capacity. Case A. T.
had a calcium-binding capacity approaching zero initially and no drug was effective in raising it.
A shift of 10 mEq. per twenty-four hours with a treatment was considered significant.

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DISCUSSION
Table I11 gives values in milliequivalents per gram of urinary phosphorus which are of
interest. The reason for this calculation is that the titration may result from the behaviour
of some complex of the phosphorus and, if so, the measure of binding capacity should be
proportional to the phosphorus content. The range of milliequivalent capacity per gram of
phosphorus is about twofold and supports the possibility that phosphorus is a factor in
solubilising calcium. The adjunct component (amino acids ?) is fluctuating independently.
Table I11 gives values for milliequivalents of capacity per milliequivalent of urinary calcium.
These are a test of the method and range about threefold. The titration gives a figure in univalent
terms expressed as milliequivalents. Consequently, if the titration is measuring a property
connected with the behaviour of the calcium then the calcium when expressed in the same units
should bear a reasonable relation to it. It is especially interesting that the two values are in the
same order of magnitude, i.e., 1 mEq. of calcium is associated in the urine with 1.5 to 5-5 mEq.
of potential calcium-binding capacity. The binding capacity is obtained with an excess of
calcium present and the milliequivalent per milliequivalent calcium figures are an index of the
equilibrium conditions for Equation 1.
Urine from over forty patients with calculous disease as well as normal persons has been
examined for calcium-binding capacity. No definite conclusions can be made except that very
severe stone-forming patients exhibit very low calcium-binding capacities ; eight out of the ten
lowest values were found in patients with multiple, bilateral, rapidly growing stones.
The role of calcium-binding capacity in stone formation fits into a scheme best outlined in
three stages. The first stage denotes molecular or ionic solutions wherein is a mixture of the
strongly electrovalent ions, the slightly dissociated organic acids, and high molecular weight
substances such as traces of proteins. The second stage includes, in addition, small physical
particles within and above the ordinary colloidal dimensions which have been termed microliths.
The particles of the second stage may be stabilised by two systems, protective colloids and
calcium-solubilising compounds. Since the microliths can grow by aggregation or by apposition
into calculi which is the third stage, then failure of either of the two stabilising systems results
in stone formation. In contrast there are certain well-known circumstances which frequently
lead to calculus formation. Elevated urinary excretion of calcium, phosphorus, cystine, and
uric acid is able to exceed in some conditions the stabilising systems.
SUMMARY

Addition of neutral calcium chloride to urine causes a decrease in pH. This decrease can
be titrated and calculated as milliequivalents of calcium-binding capacity.
The calcium-binding capacity possibly is related to the phosphorus content and is of the
same order of magnitude as the calcium content. This calcium-binding capacity may be significant
in preventing the formation of calcium-containing stones and determination of this capacity
may be helpful in equating efficacy of certain drugs used to increase calcium solubilisation.

REFERENCES

R. H., and BUTT,A. J. (1955). Clin. Chem., 1,241.

LEONARD,
MARTEL,
A. E., and CALVIN,M. (1952). Chemistry of the Metal Chelate Compounds.
(New York : Prentice Hall.)
RAAFLAUB,
J. (1956). Meth. biochem. Anal., 3, 301.
VERMEULEN,
C. W., MILLER,
G. H., and CHAPMAN,
W. H. (1956). J. UroE., 75, 592.