British Journal of Urology (lY78). 50, 442-448
A New Urinary Test for Stone "Activity" P. C. HALLSON and G. ALAN ROSE St Peter's Hospitals and Institute of Urology, London
Summary-Rapid evaporation of urine to osmolarity 1 2 0 0 results in a high incidence of envelope Wedellite and calcium phosphate crystals. The Wedellite crystals closely resemble those seen in untreated urine samples of stone formers. The incidence of crystalluria produced by these tests is higher in the stone formers than in the normal subjects, reduced by thiazides and increased by cellulose phosphate; combined thiazide and cellulose phosphate therapy was most effective in reducing crystalluria. Simple calcium and oxalate concentration products were calculated and did not correlate well with incidence of calcium oxalate crystalluria. Although the product is important, inhibitors of crystal formation must be equally important. It is postulated, but not proven, that the evaporation tests may indicate normal subjects at risk t o stone formation when exposed t o chronic dehydration and whether a stone former is still metabolically active.
One of the greatest problems in assessing the clinical status of a urinary stone former is to decide whether or not he is an active stone former at the time of evaluation. The fact that a patient has recently passed a stone gives no indication of when that stone was made. It could have been of very recent manufacture or alternatively it could have been made decades earlier when the urine composition was very different from those recently analysed. There is therefore an urgent need for a test which will assess the tendency of a given urine sample to induce stone formation. Various attempts to devise such a test have been made but have been fraught with problems. Hallson and Rose (1 976) studied urinary crystals at 37°C and found differences between random samples from normal subjects and from stone formers. However, stone formers are invariably advised to increase fluid intake, with the result that many urine samples are too dilute to show crystals even though they might have done so had fluid intake not been increased. Consequently, while the observation of crystals and crystal aggregation is of value, often no crystals are to be seen and testing is then of no particular value except to reveal that the urine sample is dilute. Our first attempt to overcome this problem was to add calcium and oxalate solutions to urine
samples as others have done (Vermeulen et al., 1964), but by means of motorised syringes and then to observe the crystals which resulted. It was thought that there might be a difference in the crystal types induced in normal subjects as opposed to stone formers. However, using this technique the calcium oxalate crystals formed did not resemble those seen in untreated urine. Instead there was a high incidence of branched crystals at slow rates of infusion and of amorphous crystals at high rates of infusion. This technique was therefore abandoned in favour of another approach which we believe has not been previously reported. The new technique was to observe all urine samples at a standard concentration by simply evaporating rapidly in a vacuum rotatory evaporator at 37°C to a urinary concentration of 1200 mmol/l and then to observe the crystals in the urine. Patients and Methods
Urine samples were collected from normal subjects and from patients with urinary calcium oxalate/phosphate stones and with idiopathic hypercalciuria. The stone formers were usually attending a metabolic stone clinic held during the morning, and some were untreated or treated with diet only, and some were on either a thiazide diuretic and/or cellulose phosphate in order to lower Read at the 34th Annual Meeting of the British Association of urinary calcium. Samples of urine were passed directly into warmed vacuum flasks and quickly Urological Surgeons in Brighton, June 1978. 442
A NEW URINARY TEST FOR STONE “ACTIVITY”
443
Initially this test was performed only at the origexamined for crystals by warm stage microscope as previously described (Hallson and Rose, 1976). If inal pH of the urine, but in view of the high incicrystals were not present then the osmolarity was dence of phosphate crystals observed at pH 6 and measured by freezing point depression technique over, and in view of our special interest in oxalate and the amount of water to be removed to yield a crystals, later urine samples were split into 2 porconcentration of 1200 mmol/l was calculated. The tions. One portion was brought to pH 5.3 before sample, usually about 25 ml, was transferred to the evaporation and the other portion was kept at the rotatory evaporator and the water removed at initial pH. Urine samples were analysed for calcium by the 37°C. By collecting the distilled water into a measuring cylinder the volume of water removed flame atomic absorption method, oxalate by the from the urine could be continuously monitored enzymatic method of Hallson and Rose (1974) and the procedure stopped at the appropriate and phosphate by Coulter Kern-0-Lab discrete time. Using this technique the rate of water multi-channel analyser using the Fiske Subbarow removed was about 2 ml per min so that the time method. required for this step was quite brief. The concentrated urine was allowed to stand for 1 h, after which it was re-examined for crystals on the 37°C Results The incidence of oxalate and phosphate crystals microscope stage (Fig. 1). occurring at natural pH before and after evaporation in urine samples from normal subjects and from stone formers without and with treatment is shown in Figure 2. Similar data largely from the same urine samples but after adjustment of urine pH to 5.3 are shown in Figure 3. It can be seen that at natural pH the incidence of crystals in unevaporated urine samples was 16% in normal subjects and 27% in the stone formers, these values being rather similar to those previously reported (Hallson and Rose, 1976). However, after evaporation of those samples which did not show crystals previously, the total incidence of crystals rose to 49.5% and 87.5% respectively. The Table shows the mean calcium and oxalate concentrations and the product of the 2 concentrations in the various groups of urine samples. The relationship of the calcium and oxalate concentration product to the occurrence or absence of oxalate crystals is shown in Figure 4 and to the incidence of oxalate crystals in Figure 5. In Figure 6 an attempt has been made to select a group of stone formers such that the mean and distribution of the calcium and oxalate concentration products match those of the normal controls. To do this the products from the normal controls were divided into 7 classes according to numerical value and the arithmetic mean of each class was found. The products for stone formers were also divided into the same 7 classes. Individual product Fig. 1 Example of envelope Wedellite (calcium oxalate dihy- values were then selected from each stone former’s drate) crystals induced by evaporating a urine sample of a stone class in such a way as to give as near as possible the former at pH 5.3. Red blood cells are also present and serve to same arithmetic mean as in the corresponding indicate size of crystals. The large aggregate (lower left) is a mass of envelope crystals which cannot all be focused simul- normal control class. Similarly the number of individual products selected from each stone formtaneously.
444
BRITISH JOURNAL OF UROLOGY p H 5.3
Natural p H
NO crystals
NO crysta~s Oxalate cr>stnls
Oxalate crystals
Pho\ph,itc cr)st,il\
Phosphate crystals
Oxalate and phosphate crystals
Oxalate and phosphate crystals
-
After evaporation Before evaporation
lormals Untreated Thiazide Cellulose Cellulose treated phosphate phosphate
treated
and thiazide treated
Stone formers
iormals Untreated Thiazide Cellulose treated
-
Cellulose
phosphate phosphate treated
and thiazide
Stone formers
Fig. 2 Incidences of crystals in urine samples at natural pH before and after evaporation from normal subjects and stone formers with idiopathic hypercalciuria.
Fig. 3 Incidences of crystals in urine samples at pH 5.3 before and after evaporation from normal subjects and stone formers with idiopathic hypercalciuria.
ing class was arranged so that the overall frequency in the stone forming distribution matched the frequency in the normal class as far as possible. The selection was made “blind” with respect to the presence or absence of crystals in the individual samples. Out of 59 samples of stone formers, 26 were removed.
be analagous to the concentration of urine in the renal tubules by water removal. It is therefore suggested that this rapid evaporation technique is preferable in studying crystal formation to techniques of addition of calcium and oxalate, since the latter give quite different crystal types. It was hoped initially that a clear difference might emerge between urine samples of normal subjects on the one hand and stone formers on the other hand. However, such a hope is perhaps rather naive for various reasons. Firstly, the evaporation technique tests what would happen to the urines were the individuals to be subjected to conditions of dehydration and there could be amongst the normal group a number of individuals who would in fact become stone formers under such conditions. Secondly, it is not certain that all the untreated stone formers were still metabolically active and some of them might have been normal at the time of the test. Hence while the incidence of
Discussion It was encouraging to have found a technique which produced crystals in urine of a structure similar to those which occur naturally in urine. The example shown in Figure 1 reveals that the envelope type of calcium oxalate crystal, believed to be the dihydrate, can be produced both as single crystals and in aggregates as occur in stone formers (Robertson et al., 1969). Such an encouraging finding is, however, not too surprising since the procedure of rapid evaporation at 37°C seems to
445
A NEW URINARY TEST FOR STONE "ACTIVITY"
Table Mean Calcium and Oxalate Concentrations and the Product of the 2 in Various Types of Urine Samples after Evaporation Group
PH
Type of crystal formed
[Cal mmolll
[Ox1 mmolll
[Cal [Ox1
N
Normals Normals
Original Original
6.01 5.32
0.283 0.299
1.80 1.58
17 65
Normals Normals
5.3 5.3
Oxalate None or phosphate only Oxalate None or phosphate only
6.19 5.46
0.297 0.285
1.86 1.56
14 81
Oxalate None or phosphate only
7.43
0.230
1.80
7
6.66
0.321
2.15
25
Oxalate None or phosphate only
8.69
0.291
2.65
8
7.52
0.307
2.36
18
Oxalate None or phosphate only
5.55
0.255
1.38
7
5.77
0.304
1.66
16
6.36
0.225
1.43
7
5.3
Oxalate None or phosphate only
5.74
0.282
1.55
13
Original
Oxalate
6.72
0.556
3.69
3
Original
None or phosphate only
5.66
0.422
2.13
7
5.3
Oxalate
8.48
0.452
3.57
5
5.3
None or phosphate only
5.35
0.550
2.38
3
Untreated stone formers Untreated stone formers Untreated stone formers Untreated stone formers Thiazide treated stone formers Thiazide treated stone formers Thiazide treated stone formers Thiazide treated stone formers Cellulose phosphate treated stone formers Cellulose phosphate treated stone formers Cellulose phosphate treated stone fromers Cellulose phosphate treated stone formers
Original Original 5.3 5.3
Original Original 5.3
crystalluria was certainly higher in stone formers than in normal subjects, it is not surprising that as shown in Figures 2 and 3 the differences between the normal subjects and the untreated stone formers were not clear cut. Two questions are posed by these results. Firstly, does the presence of crystals in evaporated normal urine samples indicate those normal subjects who would make stones under conditions of chronic dehydration? Secondly, does the absence of crystals in the stone formers indicate that they are no longer metabolically active? If these questions could be answered affirmatively it would in fact be postulated that the evaporation test really does distinguish quite clearly the stone formers from the normal subjects and that the reasons for this not being obvious is that individuals have been allocated to the wrong groups. Such a postulate is clearly not proven; much more evidence will be
required before it can be substantiated or refuted and it is put forward only as a working hypothesis for further testing. Nevertheless, regardless of whether or not the postulate is accepted, the evaporation technique can still be used as a clinical test. Thus if 2 samples from a single untreated stone former were tested, the probability of not finding crystals in either sample would fall to 1.8%.It is therefore suggested that a test for active stone formation might be based upon evaporation of 2 urine samples. If neither of these showed crystals then the subject would not be at risk of stone formation. However, under these conditions 72% of normal subjects would also show crystals, so that the positive findings would not be a distinguishing feature in this test. The response to this test of urine samples from stone formers undergoing treatment with thiazide diuretics and/or cellulose phosphate is of interest.
446
BRITISH JOURNAL OF UROLOGY 4
-
0 -X
-
3
K
m
+
2 $ 'C
2
1
N=l
.1 Normals
-tone 3
one
Formers Untreated
Stone Formers Thiazide
I ,h
Formers Cell Phos.
Natural pH
Normals
+
n 5 I
tolle Formers 1Jntrcated
Stone Formers Thiazide
tone Formers Cell Phos
Urinc at p t i 5.3
Fig. 4 The calcium X oxalate concentration products in evaporated urine samples from normal subjects and thc \tone formcrs with (+) and without ( - ) oxalate crystalluria.
It is seen in Figure 2 that thiazide increases the incidence of samples without crystals by a factor of 2, whereas cellulose phosphate increases the incidence of crystal in urine to 1 00%,a most surprising result with a therapy which is intended to reduce stone formation. However, it is to be noted that the increase in crystal formation with cellulose phosphate is mainly in the post-evaporation samples and patients are, of course, advised to drink water rather than to restrict it. The test seems to indicate that it might be particularly dangerous for patients on cellulose phosphate therapy to allow their urine to become concentrated, say by exposure to a hot environment and failure to drink sufficient water. The explanation of these findings is presumably that although cellulose phosphate lowers urinary calcium (Dent ef a f . , 1964), it also lowers urinary magnesium (Dent et al., 1964; Teotia et a1 ., 1974) and raises urinary oxalate (Marshall and Barry. 1973; Hallson eraf., 1977) and phosphate (Dent ef a f . , 1964). Combined treatment with thiazide and cellulose phosphate resulted in an increase of crystal-free urine samples to the normal value and
this treatment would therefore seem t o be advantageous. The results o f testing the urine samples at pH 5.3 are shown in Figure 3. Under these conditions phosphate crystals are greatly reduced and oxalate crystals increased, so that such a test might be considered helpful if one believes that oxalate is more important than phosphate with regard to thc common "metabolic calcium oxalate/phosphate" stones. At pH 5.3 crystal formation is again least common in the normal subjects but still occurs in 20%, is more common in untreated stone formers (53.5%). rises slightly with treatment with thiazide and more with cellulose phosphate (70%). Again the combination of thiazide and cellulose phosphate is the most effective therapy in reducing urinary crystals, and with this treatment the incidence was zero before evaporation and 200% after evaporation. The formation of crystals of a chemical in urine is only possible if the solubility (product) of the chemical is exceeded. Even then crystalluria may be prevented by inhibitors of crystal formation and
447
A NEW URINARY TEST FOR STONE "ACTIVITY"
Phosphate 4- Oxalate crystals Oulate crystals 7
4.
Phosphate crjstals
x3
0
X
.
'
2
zo 6
Normal Untreated 5 & 6 Thiazide Treated I & 8 Cellulose Phosphate Treated 0 pH 5.3 x Natural p H 1& 2 3 4
20 40 60 lncidence of Oxalate Crystals After Evaporation
Fig. 5 Mean post-evaporation urinary calcium and oxalate concentration product plotted against incidence of oxalate crystals in the same samples in various conditions. X = natural pH; 0 = pH 5.3; 1 and 2 are normal subjects and the rest are stone formers; 3 and 4 untreated; 5 and 6 treated with thiazide; 7 and 8 treated with cellulose phosphate.
of crystal growth. In this study measurements were made of urinary concentration of calcium, phosphate and oxalate and it is of some interest to see whether or not crystalluria was linked with mineral composition or whether the role of inhibitors was more important. In Figure 4 the products of calcium and oxalate concentration, henceforward referred to as [Ca Ox], are compared in urine samples with and without crystals respectively. It can be seen that the product is higher in stone formers than in normal subjects and lowered by thiazide and raised by cellulose phosphate, and it has been shown that all these differences are statistically significant. These results suggest that, as one might suppose, the concentration of calcium and oxalate are important factors in stone formation. However, there is a lack of correlation between the incidence of crystal formation and [Ca Ox] and this can be shown in several ways. Firstly, although Figure 4 shows that in normal subjects crystal formation was associated with a higher product than was absence of crystal, the reverse
-N = [Cal [ O \ ] = Phosphate rn mol/L = pH =
1.61
33 1.55
26.17
15.6X
5.3
5.3
95
Fig. 6 Incidence of phosphate and oxalate crystals among normal controls and stone formers with matched means and distribution of calcium and oxalate concentration products (see text).
was true for untreated stone formers at normal pH and for stone formers treated with thiazide and with urine of pH 5.3. However, these differences are not statistically significant and when the products for urine with and without crystals in each group of Figure 4 are compared, there are no statistically significant differences. Secondly, in Figure 5, where incidence of oxalate crystals in various groups is plotted against [Ca Ox], the correlation is remarkably poor. Thirdly, Figure 6 shows that the differences in incidences of crystals at pH 5.3 between normal subjects and stone formers does not depend upon the differences in [Ca Ox].It is thgrefore concluded that although [Ca Ox] is raised in stone formers and altered by treatment, the differences in incidence of crystal formation are not explained solely by those changes, with the possible exception that the very
448 large changes in urinary chemical composition induced by cellulose phosphate may be an important cause of the increased crystal incidence associated with this drug. In all other cases the changes in urine mineral composition do not account for changes in crystalluria and it is therefore necessary to postulate that the inhibitors of crystal formation play an important role in determining whether or not a urine will show crystal formation.
References Dent, C. E., Harper, C. M. and Parfitt, A. M. (1964). The effect of cellulose phosphate on calcium metabolism in patients with hypercalciuria. Clinical Science, 27, 41 7-425. Hallson, P. C., Kasidas, G. P. and Rose, G. A. (1977). Urinary oxalate in summer and winter in normal subjects and in stone-forming patients with idiopathic hypercalciuria, both treated and untreated with thiazide and/or cellulose phosphate. Urological Research, 4, 169-173. Hallson, P. C. and Rose, G. A. (1974). A simplified and rapid enzymatic method for determination of urinary oxalate. CIinica Chimica Acta, 55, 29-39.
JOURNAL OF UROLOGY
Hallson, P. C. and Rose, G. A. (1976). Crystalluria in normal subjects and in stone formers with and without thiazide and cellulose phosphate treatment. British Journal of Urology, 48, 515-524. Marshall, R. W. and Berry, H. (1973). Urine saturation and the formation of calcium-containing renal calculi; the effects of various forms of therapy. In "Urinary Calculi". Proceedings of the International Symposium on Renal Stone Research, Madrid, 1972. pp. 164-169. Basel: Karger. Robertson, W. G., Peacock, M. and Nordin, B. E. C. ( 1969). Calcium crystalluria in recurrent renal-stone formers. Lancet, 2, 21-24. Teotia, M., Teotia, S. P., Singh, R. K. and Teotia, N. P. ( 1974). Treatment of idiopathic hypercalciuria and nephrolithiasis with sodium cellulose phosphate. Journal of h e Association of Physicians of India, 22, 709-7 14. Vermeulen, C. W., Lyon, E. S.and Gill, W. B. (1 964). Artificial urinary concretions. Investigative Urology, I , 370-386.
The Authors P. C. Hallson, BSc, PhD, Biochemist, Institute of Urology. G. A. Rose, DM, FRCP, FRCPath, FRIC, Consultant Chemical Pathologist to st. Peter's Hospital. Requests for reprints to: G. Alan Rose, St. Paul's Hospital, Endell Street, London WC2H 9RE.
A New Urinary Test for Stone "Activity" P. C. HALLSON and G. ALAN ROSE St Peter's Hospitals and Institute of Urology, London
Summary-Rapid evaporation of urine to osmolarity 1 2 0 0 results in a high incidence of envelope Wedellite and calcium phosphate crystals. The Wedellite crystals closely resemble those seen in untreated urine samples of stone formers. The incidence of crystalluria produced by these tests is higher in the stone formers than in the normal subjects, reduced by thiazides and increased by cellulose phosphate; combined thiazide and cellulose phosphate therapy was most effective in reducing crystalluria. Simple calcium and oxalate concentration products were calculated and did not correlate well with incidence of calcium oxalate crystalluria. Although the product is important, inhibitors of crystal formation must be equally important. It is postulated, but not proven, that the evaporation tests may indicate normal subjects at risk t o stone formation when exposed t o chronic dehydration and whether a stone former is still metabolically active.
One of the greatest problems in assessing the clinical status of a urinary stone former is to decide whether or not he is an active stone former at the time of evaluation. The fact that a patient has recently passed a stone gives no indication of when that stone was made. It could have been of very recent manufacture or alternatively it could have been made decades earlier when the urine composition was very different from those recently analysed. There is therefore an urgent need for a test which will assess the tendency of a given urine sample to induce stone formation. Various attempts to devise such a test have been made but have been fraught with problems. Hallson and Rose (1 976) studied urinary crystals at 37°C and found differences between random samples from normal subjects and from stone formers. However, stone formers are invariably advised to increase fluid intake, with the result that many urine samples are too dilute to show crystals even though they might have done so had fluid intake not been increased. Consequently, while the observation of crystals and crystal aggregation is of value, often no crystals are to be seen and testing is then of no particular value except to reveal that the urine sample is dilute. Our first attempt to overcome this problem was to add calcium and oxalate solutions to urine
samples as others have done (Vermeulen et al., 1964), but by means of motorised syringes and then to observe the crystals which resulted. It was thought that there might be a difference in the crystal types induced in normal subjects as opposed to stone formers. However, using this technique the calcium oxalate crystals formed did not resemble those seen in untreated urine. Instead there was a high incidence of branched crystals at slow rates of infusion and of amorphous crystals at high rates of infusion. This technique was therefore abandoned in favour of another approach which we believe has not been previously reported. The new technique was to observe all urine samples at a standard concentration by simply evaporating rapidly in a vacuum rotatory evaporator at 37°C to a urinary concentration of 1200 mmol/l and then to observe the crystals in the urine. Patients and Methods
Urine samples were collected from normal subjects and from patients with urinary calcium oxalate/phosphate stones and with idiopathic hypercalciuria. The stone formers were usually attending a metabolic stone clinic held during the morning, and some were untreated or treated with diet only, and some were on either a thiazide diuretic and/or cellulose phosphate in order to lower Read at the 34th Annual Meeting of the British Association of urinary calcium. Samples of urine were passed directly into warmed vacuum flasks and quickly Urological Surgeons in Brighton, June 1978. 442
A NEW URINARY TEST FOR STONE “ACTIVITY”
443
Initially this test was performed only at the origexamined for crystals by warm stage microscope as previously described (Hallson and Rose, 1976). If inal pH of the urine, but in view of the high incicrystals were not present then the osmolarity was dence of phosphate crystals observed at pH 6 and measured by freezing point depression technique over, and in view of our special interest in oxalate and the amount of water to be removed to yield a crystals, later urine samples were split into 2 porconcentration of 1200 mmol/l was calculated. The tions. One portion was brought to pH 5.3 before sample, usually about 25 ml, was transferred to the evaporation and the other portion was kept at the rotatory evaporator and the water removed at initial pH. Urine samples were analysed for calcium by the 37°C. By collecting the distilled water into a measuring cylinder the volume of water removed flame atomic absorption method, oxalate by the from the urine could be continuously monitored enzymatic method of Hallson and Rose (1974) and the procedure stopped at the appropriate and phosphate by Coulter Kern-0-Lab discrete time. Using this technique the rate of water multi-channel analyser using the Fiske Subbarow removed was about 2 ml per min so that the time method. required for this step was quite brief. The concentrated urine was allowed to stand for 1 h, after which it was re-examined for crystals on the 37°C Results The incidence of oxalate and phosphate crystals microscope stage (Fig. 1). occurring at natural pH before and after evaporation in urine samples from normal subjects and from stone formers without and with treatment is shown in Figure 2. Similar data largely from the same urine samples but after adjustment of urine pH to 5.3 are shown in Figure 3. It can be seen that at natural pH the incidence of crystals in unevaporated urine samples was 16% in normal subjects and 27% in the stone formers, these values being rather similar to those previously reported (Hallson and Rose, 1976). However, after evaporation of those samples which did not show crystals previously, the total incidence of crystals rose to 49.5% and 87.5% respectively. The Table shows the mean calcium and oxalate concentrations and the product of the 2 concentrations in the various groups of urine samples. The relationship of the calcium and oxalate concentration product to the occurrence or absence of oxalate crystals is shown in Figure 4 and to the incidence of oxalate crystals in Figure 5. In Figure 6 an attempt has been made to select a group of stone formers such that the mean and distribution of the calcium and oxalate concentration products match those of the normal controls. To do this the products from the normal controls were divided into 7 classes according to numerical value and the arithmetic mean of each class was found. The products for stone formers were also divided into the same 7 classes. Individual product Fig. 1 Example of envelope Wedellite (calcium oxalate dihy- values were then selected from each stone former’s drate) crystals induced by evaporating a urine sample of a stone class in such a way as to give as near as possible the former at pH 5.3. Red blood cells are also present and serve to same arithmetic mean as in the corresponding indicate size of crystals. The large aggregate (lower left) is a mass of envelope crystals which cannot all be focused simul- normal control class. Similarly the number of individual products selected from each stone formtaneously.
444
BRITISH JOURNAL OF UROLOGY p H 5.3
Natural p H
NO crystals
NO crysta~s Oxalate cr>stnls
Oxalate crystals
Pho\ph,itc cr)st,il\
Phosphate crystals
Oxalate and phosphate crystals
Oxalate and phosphate crystals
-
After evaporation Before evaporation
lormals Untreated Thiazide Cellulose Cellulose treated phosphate phosphate
treated
and thiazide treated
Stone formers
iormals Untreated Thiazide Cellulose treated
-
Cellulose
phosphate phosphate treated
and thiazide
Stone formers
Fig. 2 Incidences of crystals in urine samples at natural pH before and after evaporation from normal subjects and stone formers with idiopathic hypercalciuria.
Fig. 3 Incidences of crystals in urine samples at pH 5.3 before and after evaporation from normal subjects and stone formers with idiopathic hypercalciuria.
ing class was arranged so that the overall frequency in the stone forming distribution matched the frequency in the normal class as far as possible. The selection was made “blind” with respect to the presence or absence of crystals in the individual samples. Out of 59 samples of stone formers, 26 were removed.
be analagous to the concentration of urine in the renal tubules by water removal. It is therefore suggested that this rapid evaporation technique is preferable in studying crystal formation to techniques of addition of calcium and oxalate, since the latter give quite different crystal types. It was hoped initially that a clear difference might emerge between urine samples of normal subjects on the one hand and stone formers on the other hand. However, such a hope is perhaps rather naive for various reasons. Firstly, the evaporation technique tests what would happen to the urines were the individuals to be subjected to conditions of dehydration and there could be amongst the normal group a number of individuals who would in fact become stone formers under such conditions. Secondly, it is not certain that all the untreated stone formers were still metabolically active and some of them might have been normal at the time of the test. Hence while the incidence of
Discussion It was encouraging to have found a technique which produced crystals in urine of a structure similar to those which occur naturally in urine. The example shown in Figure 1 reveals that the envelope type of calcium oxalate crystal, believed to be the dihydrate, can be produced both as single crystals and in aggregates as occur in stone formers (Robertson et al., 1969). Such an encouraging finding is, however, not too surprising since the procedure of rapid evaporation at 37°C seems to
445
A NEW URINARY TEST FOR STONE "ACTIVITY"
Table Mean Calcium and Oxalate Concentrations and the Product of the 2 in Various Types of Urine Samples after Evaporation Group
PH
Type of crystal formed
[Cal mmolll
[Ox1 mmolll
[Cal [Ox1
N
Normals Normals
Original Original
6.01 5.32
0.283 0.299
1.80 1.58
17 65
Normals Normals
5.3 5.3
Oxalate None or phosphate only Oxalate None or phosphate only
6.19 5.46
0.297 0.285
1.86 1.56
14 81
Oxalate None or phosphate only
7.43
0.230
1.80
7
6.66
0.321
2.15
25
Oxalate None or phosphate only
8.69
0.291
2.65
8
7.52
0.307
2.36
18
Oxalate None or phosphate only
5.55
0.255
1.38
7
5.77
0.304
1.66
16
6.36
0.225
1.43
7
5.3
Oxalate None or phosphate only
5.74
0.282
1.55
13
Original
Oxalate
6.72
0.556
3.69
3
Original
None or phosphate only
5.66
0.422
2.13
7
5.3
Oxalate
8.48
0.452
3.57
5
5.3
None or phosphate only
5.35
0.550
2.38
3
Untreated stone formers Untreated stone formers Untreated stone formers Untreated stone formers Thiazide treated stone formers Thiazide treated stone formers Thiazide treated stone formers Thiazide treated stone formers Cellulose phosphate treated stone formers Cellulose phosphate treated stone formers Cellulose phosphate treated stone fromers Cellulose phosphate treated stone formers
Original Original 5.3 5.3
Original Original 5.3
crystalluria was certainly higher in stone formers than in normal subjects, it is not surprising that as shown in Figures 2 and 3 the differences between the normal subjects and the untreated stone formers were not clear cut. Two questions are posed by these results. Firstly, does the presence of crystals in evaporated normal urine samples indicate those normal subjects who would make stones under conditions of chronic dehydration? Secondly, does the absence of crystals in the stone formers indicate that they are no longer metabolically active? If these questions could be answered affirmatively it would in fact be postulated that the evaporation test really does distinguish quite clearly the stone formers from the normal subjects and that the reasons for this not being obvious is that individuals have been allocated to the wrong groups. Such a postulate is clearly not proven; much more evidence will be
required before it can be substantiated or refuted and it is put forward only as a working hypothesis for further testing. Nevertheless, regardless of whether or not the postulate is accepted, the evaporation technique can still be used as a clinical test. Thus if 2 samples from a single untreated stone former were tested, the probability of not finding crystals in either sample would fall to 1.8%.It is therefore suggested that a test for active stone formation might be based upon evaporation of 2 urine samples. If neither of these showed crystals then the subject would not be at risk of stone formation. However, under these conditions 72% of normal subjects would also show crystals, so that the positive findings would not be a distinguishing feature in this test. The response to this test of urine samples from stone formers undergoing treatment with thiazide diuretics and/or cellulose phosphate is of interest.
446
BRITISH JOURNAL OF UROLOGY 4
-
0 -X
-
3
K
m
+
2 $ 'C
2
1
N=l
.1 Normals
-tone 3
one
Formers Untreated
Stone Formers Thiazide
I ,h
Formers Cell Phos.
Natural pH
Normals
+
n 5 I
tolle Formers 1Jntrcated
Stone Formers Thiazide
tone Formers Cell Phos
Urinc at p t i 5.3
Fig. 4 The calcium X oxalate concentration products in evaporated urine samples from normal subjects and thc \tone formcrs with (+) and without ( - ) oxalate crystalluria.
It is seen in Figure 2 that thiazide increases the incidence of samples without crystals by a factor of 2, whereas cellulose phosphate increases the incidence of crystal in urine to 1 00%,a most surprising result with a therapy which is intended to reduce stone formation. However, it is to be noted that the increase in crystal formation with cellulose phosphate is mainly in the post-evaporation samples and patients are, of course, advised to drink water rather than to restrict it. The test seems to indicate that it might be particularly dangerous for patients on cellulose phosphate therapy to allow their urine to become concentrated, say by exposure to a hot environment and failure to drink sufficient water. The explanation of these findings is presumably that although cellulose phosphate lowers urinary calcium (Dent ef a f . , 1964), it also lowers urinary magnesium (Dent et al., 1964; Teotia et a1 ., 1974) and raises urinary oxalate (Marshall and Barry. 1973; Hallson eraf., 1977) and phosphate (Dent ef a f . , 1964). Combined treatment with thiazide and cellulose phosphate resulted in an increase of crystal-free urine samples to the normal value and
this treatment would therefore seem t o be advantageous. The results o f testing the urine samples at pH 5.3 are shown in Figure 3. Under these conditions phosphate crystals are greatly reduced and oxalate crystals increased, so that such a test might be considered helpful if one believes that oxalate is more important than phosphate with regard to thc common "metabolic calcium oxalate/phosphate" stones. At pH 5.3 crystal formation is again least common in the normal subjects but still occurs in 20%, is more common in untreated stone formers (53.5%). rises slightly with treatment with thiazide and more with cellulose phosphate (70%). Again the combination of thiazide and cellulose phosphate is the most effective therapy in reducing urinary crystals, and with this treatment the incidence was zero before evaporation and 200% after evaporation. The formation of crystals of a chemical in urine is only possible if the solubility (product) of the chemical is exceeded. Even then crystalluria may be prevented by inhibitors of crystal formation and
447
A NEW URINARY TEST FOR STONE "ACTIVITY"
Phosphate 4- Oxalate crystals Oulate crystals 7
4.
Phosphate crjstals
x3
0
X
.
'
2
zo 6
Normal Untreated 5 & 6 Thiazide Treated I & 8 Cellulose Phosphate Treated 0 pH 5.3 x Natural p H 1& 2 3 4
20 40 60 lncidence of Oxalate Crystals After Evaporation
Fig. 5 Mean post-evaporation urinary calcium and oxalate concentration product plotted against incidence of oxalate crystals in the same samples in various conditions. X = natural pH; 0 = pH 5.3; 1 and 2 are normal subjects and the rest are stone formers; 3 and 4 untreated; 5 and 6 treated with thiazide; 7 and 8 treated with cellulose phosphate.
of crystal growth. In this study measurements were made of urinary concentration of calcium, phosphate and oxalate and it is of some interest to see whether or not crystalluria was linked with mineral composition or whether the role of inhibitors was more important. In Figure 4 the products of calcium and oxalate concentration, henceforward referred to as [Ca Ox], are compared in urine samples with and without crystals respectively. It can be seen that the product is higher in stone formers than in normal subjects and lowered by thiazide and raised by cellulose phosphate, and it has been shown that all these differences are statistically significant. These results suggest that, as one might suppose, the concentration of calcium and oxalate are important factors in stone formation. However, there is a lack of correlation between the incidence of crystal formation and [Ca Ox] and this can be shown in several ways. Firstly, although Figure 4 shows that in normal subjects crystal formation was associated with a higher product than was absence of crystal, the reverse
-N = [Cal [ O \ ] = Phosphate rn mol/L = pH =
1.61
33 1.55
26.17
15.6X
5.3
5.3
95
Fig. 6 Incidence of phosphate and oxalate crystals among normal controls and stone formers with matched means and distribution of calcium and oxalate concentration products (see text).
was true for untreated stone formers at normal pH and for stone formers treated with thiazide and with urine of pH 5.3. However, these differences are not statistically significant and when the products for urine with and without crystals in each group of Figure 4 are compared, there are no statistically significant differences. Secondly, in Figure 5, where incidence of oxalate crystals in various groups is plotted against [Ca Ox], the correlation is remarkably poor. Thirdly, Figure 6 shows that the differences in incidences of crystals at pH 5.3 between normal subjects and stone formers does not depend upon the differences in [Ca Ox].It is thgrefore concluded that although [Ca Ox] is raised in stone formers and altered by treatment, the differences in incidence of crystal formation are not explained solely by those changes, with the possible exception that the very
448 large changes in urinary chemical composition induced by cellulose phosphate may be an important cause of the increased crystal incidence associated with this drug. In all other cases the changes in urine mineral composition do not account for changes in crystalluria and it is therefore necessary to postulate that the inhibitors of crystal formation play an important role in determining whether or not a urine will show crystal formation.
References Dent, C. E., Harper, C. M. and Parfitt, A. M. (1964). The effect of cellulose phosphate on calcium metabolism in patients with hypercalciuria. Clinical Science, 27, 41 7-425. Hallson, P. C., Kasidas, G. P. and Rose, G. A. (1977). Urinary oxalate in summer and winter in normal subjects and in stone-forming patients with idiopathic hypercalciuria, both treated and untreated with thiazide and/or cellulose phosphate. Urological Research, 4, 169-173. Hallson, P. C. and Rose, G. A. (1974). A simplified and rapid enzymatic method for determination of urinary oxalate. CIinica Chimica Acta, 55, 29-39.
JOURNAL OF UROLOGY
Hallson, P. C. and Rose, G. A. (1976). Crystalluria in normal subjects and in stone formers with and without thiazide and cellulose phosphate treatment. British Journal of Urology, 48, 515-524. Marshall, R. W. and Berry, H. (1973). Urine saturation and the formation of calcium-containing renal calculi; the effects of various forms of therapy. In "Urinary Calculi". Proceedings of the International Symposium on Renal Stone Research, Madrid, 1972. pp. 164-169. Basel: Karger. Robertson, W. G., Peacock, M. and Nordin, B. E. C. ( 1969). Calcium crystalluria in recurrent renal-stone formers. Lancet, 2, 21-24. Teotia, M., Teotia, S. P., Singh, R. K. and Teotia, N. P. ( 1974). Treatment of idiopathic hypercalciuria and nephrolithiasis with sodium cellulose phosphate. Journal of h e Association of Physicians of India, 22, 709-7 14. Vermeulen, C. W., Lyon, E. S.and Gill, W. B. (1 964). Artificial urinary concretions. Investigative Urology, I , 370-386.
The Authors P. C. Hallson, BSc, PhD, Biochemist, Institute of Urology. G. A. Rose, DM, FRCP, FRCPath, FRIC, Consultant Chemical Pathologist to st. Peter's Hospital. Requests for reprints to: G. Alan Rose, St. Paul's Hospital, Endell Street, London WC2H 9RE.
