Clinica Chimica Acta, 124 (1982) 149-155

Elsevier Biomedical Press

149

CCA 2243

Calcium oxalate nephrolithiasis: an easy way to

detect an imbalance between promoting and
inhibiting factors
B. Baggio, G. Gambaro, 0. Oliva, S. Favaro and A. Borsatti *
Clinica Mediea I, Vniuersitb di Padooa, 35100 Padooa (Italyl
(Received February 2nd; revision May 11 th, 1982)

Summary

Calcium oxalate stone formation depends on both urine oversaturation with

calcium oxalate - which in turn depends mainly on oxalate excretion - and the
excretion of inhibitors; the possibility that a ratio of these variables might differentiate stone-formers from stone-free subjects was explored. 24-h urine samples from 20
control subjects and 53 idiopathic calcium oxalate stone-formers receiving a standard diet were studied. A further group of 2-h urine samples (from 7 to 9 a.m.),
collected after an overnight fast from 16 non-stone and 24 stone-forming persons on
a normal diet, were also examined. The ratio oxalate/citrate X acid mucopolysaccharides (Ox/Cit X AMPS) seems capable of differentiating more than 80% of
stone-formers from non-stone-formers using both 24- and 2-h urine collection.

Introduction

Calcium oxalate nephrolithiasis is the most common type of kidney stone disease,
but despite wide investigation, its pathogenesis remains unclear. The degree of urine
oversaturation with calcium oxalate is without doubt a necessary factor, but by itself
does not explain crystal growth and aggregation, since many stone-free subjects
show urine oversaturated with calcium oxalate [ 1,2]. This observation has led first to
the hypothesis, and then to the demonstration, of some protective inhibitors in urine
[3]. It now seems reasonable to consider calcium oxalate stone disease as a multifactorial disorder with the risk of forming stones dependent upon a disturbance in the
balance between oversaturation of urine and concentration of protective inhibitors.
Many attempts have been made to detect either an excess in promoting or a
defect in inhibiting factors in the urine of stone-formers. The pertinent literature is
Correspondence:
Padova, Italy.

A. Borsatti, Clinica Medica I, Universita

0009-8981/82/000&0000/$02.75

di Padova, Policlinico

0 1982 Elsevier Biomedical

Press

Universitario,

35100

150

extremely contradictory, with much confusion arising from a chemical error. In fact,
urine constituents are usually measured in amount per day, which is quite misleading
since, from a chemical point of view, the important criterion is the concentration and
not the amount. The search for an abnormal relationship between promoting and
inhibiting factors seems to represent a more coherent approach to the problem
rather than the study of a single constituent. In fact, with this approach it makes no
difference whether concentration or amount are considered, since the relationship
remains exactly the same. If such a disturbance can be measured, then the risk of
forming stones may be estimated.
Our objective was to try to find an easy index with good sensitivity. We first
considered that if urine saturation is estimated theoretically as suggested by Marshall
and Robertson [4], then there should be a correlation with either calcium or oxalate;
if urine saturation is obtained empirically as suggested by Pak and Ohata [S]
correlation between urine saturation and ion concentration would be found [6].
Moreover, since oxalate appears more involved in urine oversaturation than calcium,
it should be more meaningful [7,8]. With respect to inhibitory substances, pyrophosphate was considered to have very weak inhibitory activity, but citrate and acid
mucopolysaccha~des (AMPs) are regarded as major inhibitors [9,10]. In the light of
these factors, any imbalance between promoting and inhibiting factors in calcium
oxalate stone disease should be demonstrable by a ratio between oxalate on the one
hand and citrate and AMPS taken alone or together on the other. The results
obtained, using this approach, are described in this paper.
Material and methods

Twenty normal subjects (10 males and 10 females) and 53 patients (28 males and
25 females) with recurrent idiopathic calcium oxalate nephrolithiasis, and who had
passed at least one stone during the two months immediately preceding our
investigation were studied. Four days before the study, both patients and controls
were placed on a standard diet containing 800 mg calcium, 7.5 mg oxalate, 85 mg
purine and 900 mg phosphate and were asked to drink 1 liter per day of mineral-free
water. A 24-h urine was collected on the 4th day. A single urine collection (from 7 to
9 a.m.) following an overnight fast was made from an additional group of 16
controls (11 males and 5 females) and from 24 stone-forming patients (14 males and
10 females) on a completely normal diet and without any restriction in water intake.
Renal function, as assessed by creatinine clearance, was normal in all patients. Urine
culture was negative in all patients. Serum PTH, urine CAMP were in the normal
range. As soon as the specimens had been collected, volume and pH were recorded.
Samples were then immediately assayed for sodium (by flame photometry), phosphate [ 111, ammonia 1121, uric acid [ 131, pyrophosphate f 141, citrate [15] and AMPS
[16]. To 100 ml urine 1 ml of concentrated HCl was then added and subsequently
calcium and magnesium were measured by atomic absorption and oxalate by the
method of Hodgkinson and Williams [ 171.Although the calorimetric assay of oxalate
has been criticized, it seems to give almost the same results as more sophisticated
procedures such as gas chromatography [IS]. Patients with a urinary excretion of the

CONCENT~TION

URINE

II

ap <O.OS; bp (0.02;

Uric acid
Oxalate
Calcium
Phosphate
Magnesium
Citrate
Pyrophosphate
AMPS
Volume

0.87
0.13
1.61
19.27
0.86
1.44
15.56
17.92
0.47

in mmo1/24

cp -=O.Ol.

3.502
0.78
0.28-c
0.11
5.04*
1.60
34.61 ;t 14.75
3.03%
0.80
3.40*
1.58
38.65 * 20.02
93.25 rt 20.7 1
1115
* 268

controls

24-h collection

as mean*SD

3.34rt
1.42
0.41*
0.15
4.20*
2.34
37.31= 19.32
2.49*
0.96
2.29*
2.69
22.562 18.07
58.19t20.97
6.012
0.54

stone-formers

0.65
4.24
0.35
1.60
0.17
1.74
2.46 b
3.62
0.98

0.36
0.025
0.33
4.15
0.21
0.35
3.63
9.84
102

controls

3.42*
0.83
0.24*
0.01
3.622
1.71
14.97f 12.20
2.04)
1.22
1.93-c 1.47
18.61~ 15.38
66.23*21.95
6.4Of
0.83

stone-formers

0.38*
0.20
0.03*
0.01
0.40*
0.20
l.81*
0.89
0.24*
0.15
0.16*
0.05
2.592
2.50
8.81*
5.09
144
--fll3

stone-former5

and volume (ml).

FACTORS

* 0.20
* 0.02
* 0.24
2 5.10
* 0.24
* 2.32
* 4.20
* 6.24
54

2-h collection

(cmol)

ANTI-LITHOGENIC

4.16-t
1.40
0.25~
0.08
3.38*
1.76
29.72k22.12
2.232
1.00
3.52k
1.24
33.632 27.48
95.95 -c 24.80
6.10-t 0.80

0.82
3.95 =
0.42
0.69
0.5 1
2.04 a
2.39 b
4.17 e
0.6 1

AND

controls

2-h collection

and pH.

(pmol/l)

FACTORS

h except AMPS (me), pyrophosphate

3.79-c
1.91
0.49-c
0.22
4.822
2.52
44.21 -t 25.15
2.97-i:
1.42
2.69*
1.55
25.21 -c 21.08
69.50-t
26.37
1197
-c 336

stone-formers

h or mmol/Z

OF THE LITHOGENIC

Cp -=O.ool.

(24 h and 2 h respectively)

dp co.005;

3.06;?:
0.26k
4.44*
33.80*
2.621t:
2.98*
33.52rt:
80.31 -c
6.10*

controls

24-h collection

pyrophosphate

AND ANTI-LITH~ENIC

except AMPS (mg/l),

OF THE LITH~ENI~

as mean* SD in mmol/l

=p co.01;

EXCRETION

bp (0.02;

All values are expressed

URINARY

TABLE

ap co.05;

Uric acid
Oxalate
Calcium
Phosphate
Magnesium
Citrate
~r~hospha~
AMPS
PH

All values are expressed

TABLE

d
a
=

0.33
0.94
1.03
2.21 a
0.83
3.91 c
0.99
0.57
1.39

0.93
0.37
0.43
2.72
0.51
3.55
2.22
3.98
0.93

g
-

152

ions greater than the mean plus two standard deviations

found in controls were
considered
to be hypercalciuric,
hyperoxaluric
or hyperuricuric.
The percent incidence of hypercalciuria
was 16%, hyperoxaluria
34% and hyperuricuria
17% in our
population
of stone-formers.
Statistical analysis was carried out using Students t test
for unpaired
variables
and Youdens
J index to assess the accuracy of the
diagnostic procedure. A J index of 0 means that the test has no diagnostic value,
whereas an index of 1 means that the test gave correct information
[ 191.
Results
Molar concentrations
of H+ , uric acid, calcium, oxalate, ammonia,
sodium,
phosphate,
magnesium,
pyrophosphate,
citrate and AMPS are reported in Table I.
Table II reports the same data referred to the total amount excreted in the 24-h and
2-h urine specimens.
Of the measured variables, molar concentration
and total amount/day
of oxalate
were higher in stone-formers
and citrate, pyrophosphate
and AMPS were significantly lower in stone-formers,
compared to controls. However, if the 2-h specimen is
considered,
the observed difference in oxalate disappears
and the stone-formers
seem to excrete less phosphate
than normals. We then challenged
the diagnostic
accuracy and sensitivity of the indices Ox/Cit, OX/AMPS and Ox/Cit X AMPS.
Ox/Cit gave a J index of 0.67, OX/AMPS an index of 0.70, and Ox/Cit X AMPS
an index of 0.82, which means that the latter separates more than 80% of the
stone-formers
from the stone-free subjects. With regard to the 2-h specimen, the J
indices for Ox/Cit
and Ox/Cit X AMPS were almost the same (0.67 and 0.83,
respectively), but the OX/AMPS ratio was less significant (0.33) (Table III).
Discussion
The pathogenesis
of CaOx nephrolithiasis
the probability
of forming stones is likely

is almost certainly multifactorial

and
to depend on a combination
of risk

TABLE III
DIAGNOSTIC

ACCURACY

OF THE SELECTED

INDICES

Index

* Normal range

False
positive

False
negative

Ox/Citrate

* 693.30+615.40
** 746.02 + 595.18

5%
0%

28%
33%

0.67
0.67

OX/AMPS
Ox/Cit

AMPS

* (Mean*2

* 27.60+
** 26.64+

23.00
14.96

0%
0%

30%
67%

0.70
0.33

* 8.81+
** 8.25+

5.78
5.44

5%
0%

13%
17%

0.82
0.83

SD)X IO4 are reported for both 24 h (*) and 2 h (**) collections.

153

factors. An attempt to quantitate this risk was reported by Robertson et al [20] who
developed a saturation-inhibition index.
However, it was difficult and time-consuming to determine both the state of urine
oversaturation and inhibitory activity. The same workers later attempted a different
approach to the problem, starting from the observation that stone-formers showed a
higher urine pH? excreted more calcium, oxalate and uric acid, and less AMPS [21].
Robertson et al calculated the frequency distribution of the single variables and used
them to quantitate the risk of stone formation. They then obtained an overall
probability of forming stones from each risk factor. In this procedure, all the
variables considered must be stochastically independent, and Robertson and Peacock

ox

Xl04

cIT./iNB

100

50

30

10

C
2 HDDRS

21HOURS

A CONTROLS
. ST@NE FORI'ERS
Fig. 1. Shows the abnormal distribution of the index Ox/Cit X AMPS in stone-formers
24-h urine collections.

using both 2-h and

154

have recently reported a correlation between calcium and oxalate in a stone-forming

population,
thus rendering this approach untenable
[8].
In an attempt to detect an imbalance between promoting and inhibiting factors in
calcium oxalate stone-formers,
the possibility
of measuring
a biochemical
steady
state through the use of a ratio or a product of the concentration
of some solutes was
considered.
To this end, it seemed reasonable
to choose oxalate as the most likely
promoting
factor since all the experimental
data stress its importance
rather than
that of calcium [7,8]. Furthermore,
we have found an higher than normal 24-h
urinary excretion of oxalate in stone-formers,
even if this difference disappears when
a 2-h urine specimen is considered.
However, since the latter collection was made
after an overnight fast, this difference could be explained by a higher than normal
enteral absorption
of oxalate in stone-formers,
as suggested by Hodgkinson
[22].
The choice of inhibitors
was more difficult, however, and as pyrophosphate
inhibitory activity has been shown to be very weak, it was discarded. It appears that
the major inhibitory
role is played by both citrate and AMPS and these two were
considered
separately and together. Three indices resulted: Ox/Cit. OX/AMPS,
and Ox/Cit X AMPS. All three provided a good differentiation
between stone-forming and stone-free subjects; however Ox/Cit X AMPS seems capable of separating more than 80% of stone-formers
from controls (see Fig. 1). In other words, more
than 80% of stone-formers
show an imbalance between stone-promoting
and stoneinhibiting
factors. From a practical point of view it is interesting
to note that the
best discriminating
index, i.e. Ox/Cit X AMPS provided almost the same results
when the 2-h specimen on a free diet was considered (see Fig. 1). This observation
suggests that the troublesome
procedure of collecting urine for 24-h and imposing
dietary restriction might be avoided.
It is clear that further study is required. However, if this index proves reliable, it
may provide a useful tool in assessing stone-forming
patients. Moreover, it may
enable better evaluation
of the effects of therapy, and finally it may represent a
meaningful
procedure
for the differentiation
of stone-formers
from the general
population.
References
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WG, Peacock M, Nordin BEC. Activity product in stone-forming
and non stone-forming
urine. Clin Sci 1968; 34: 519-594.
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WG, Hodgkinson
A, Nordin BEC. The relation between the
concentration
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155

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