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 differenti- ate 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 stan- dard 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 mucopolysac- charides’ (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 multifac- torial 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: A. Borsatti, Clinica Medica I, Universita di Padova, Policlinico Universitario, 35100

Padova, Italy.

0009-8981/82/000&0000/$02.75 0 1982 Elsevier Biomedical Press

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, pyrophos- phate 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), phos- phate [ 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

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ions greater than the mean plus two standard deviations found in controls were considered to be hypercalciuric, hyperoxaluric or hyperuricuric. The percent inci- dence of hypercalciuria was 16%, hyperoxaluria 34% and hyperuricuria 17% in our

population of stone-formers. Statistical analysis was carried out using Student’s t test for unpaired variables and Youden’s ‘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 signifi- cantly 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 is almost certainly multifactorial and the probability of forming stones is likely to depend on a combination of risk

TABLE III

DIAGNOSTIC ACCURACY OF THE SELECTED INDICES

Index * Normal range False positive

False J negative

Ox/Citrate

OX/AMPS

Ox/Cit AMPS

* 693.30+615.40 5% 28% 0.67 ** 746.02 + 595.18 0% 33% 0.67

* 27.60+ 23.00 0% 30% 0.70 ** 26.64+ 14.96 0% 67% 0.33

* 8.81+ 5.78 5% 13% 0.82 ** 8.25+ 5.44 0% 17% 0.83

* (Mean*2 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

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24-h urine collections.

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-for-

ming and stone-free subjects; however ‘Ox/Cit X AMPS’ seems capable of separat- ing 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 stone-

inhibiting 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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urine. Clin Sci 1968; 34: 519-594.

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taining renal stones. Clin Sci 1972; 43: 433-441.

3 Fleisch H. Inhibitors and promoters of stones. Kidney Int 1978; 13: 361-371.

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