Struvite infection calculi in dogs : Problems with urinary calculus identification , and the value of the results

I DENTIFYING THE CRYSTALLINE COMPONENTS of a urinary calculus is a valuable aid to correct diagnosis, and subsequent treatment of the underlying cause of the problem. Calculus formation is often an end product brought about by urine with a high concentration of insoluble material causing bladder irritation. This promotes bacterial infection by Proteus, which not only raises the pH in the bladder and generates ammonia, but also provides sites for mineral nucleation and crystal growth. Treating the infection and removing the stone will not necessarily cure the disorder. Correct identification of a calculus as struvite and even its potassium analogue can be critical in choice of curative protocols, but this is not always straightforward. Our experience with ‘problem calculi’ is described.


Introduction
We have been systematically analysing urinary calculi by the X-ray powder diffraction method since 1978, in both humans and domestic animals.l-3 The formation of a struvite calculus is normally due to the presence of ammonia and a raised pH in the urine, caused by infection.We have found over the past four years that some struvite bladder calculi from dogs display abnormal intensity peak patterns that are not immediately identifiable, even through the d-spacings of the planes appear to be generally correct. 4These discrepancies are so large as to be of significance and warrant reporting.

Experimental
X-ray powder diffraction (XRD) patterns were recorded with cobalt K α radiation (λ = 1.7889Å) as described in ref. 1.A selection of observed patterns is shown in Fig.A (see supplementary material online), and a list of the d-spacings and the relative intensities of diffraction peaks are given in Table 1 (online).The calculi were also analysed for the presence of various chemical elements using the EDAX technique on a JEOL scanning electron microscope, because the presence of calcium is indicative of amorphous hydroxyl apatite, which often exists as an amorphous solid and is therefore not evident in the XRD scan.

Results
Typical analytical data are presented in Fig. B (online).The results from a collection of struvite stones are given in Fig. 1 and Fig.A (online).A comparison of the observed peak intensities (Table 1) with the standard pattern 4 shows quite clearly that few of the samples (e.g.A-D,

The problem of potassium
The most commonly occurring mineral species in calculi from dogs is struvite, an ammonium magnesium phosphate hydrate [NH 4 MgPO 4 @6H 2 O].However, we were surprised when potassium was detected by EDAX in several of the stones.It transpires that a well-defined potassium analogue [KMgPO 4 @6H 2 O] of struvite exists, where K + replaces the NH 4 + ammonium cations.These two compounds are isostructural, 6,7 with the existence of a complete isomorphous series from 100% K + to 100% NH 4 + struvite. 6This ion replacement is possible, as the ionic radii of K + and NH 4 + are almost identical 8 (1.52 vs 1.51 Å).
It is evident that when the struvite (infection) calculus forms and is growing, any potassium present in the urine can be incorporated into the crystal structure of the stone.This potassium can be detected by EDAX, but can also show itself by subtle effect on all the d-spacings (those of ideal pure struvite are about 2% greater than a School of Earth Science, University of KwaZulu-Natal, Durban 4041, South Africa.b School of Pure and Applied Chemistry, University of KwaZulu-Natal, Durban.*Author for correspondence.E-mail: dunleveyj@ukzn.ac.za

Research in Action
those of the potassium analogue).Table 2 (see supplementary material online) compares the unit cell dimensions and important d-spacings of the two crystalline compounds.It is clear that material that contains both K + and NH 4 + cations will have d-spacings of intermediate size; but the effect is very small, with differences being detectable only in the second decimal place of the d-spacing.Unfortunately, this level of precision is not usually attainable during typical routine powder diffraction analysis of calculi samples.
Figures C and D (both online) are projections of the crystal structures along the b-axis.It is evident that the site occupied by the K + and NH 4 + ions lies on the 110, 020, 021, 200 and 130 planes and diffraction from these should be most affected by the replacement of NH 4 + by K + (Table 2).

An unusual result
Calcium is regularly found in calculi, 2,3 most commonly as the phosphate [hydroxyl apatite Ca 5 (PO 4 ) 3 (OH)] and less often as an oxalate [monohydrate whewellite CaC 2 O 4 @H 2 O; dihydrate weddelite CaC 2 O 4 @2H 2 O].The struvite calculi from the dog named Rambo consist of two types of material: (i) dark, hard, containing only magnesium (typical struvite); (ii) softer, granular containing some calcium.The intensities of the diffraction peaks from the type (ii) material were distinctly weaker than those from the type (i) material, indicating that type (ii) was less crystalline, that is, the internal structure was not as regular as in the ideal struvite structure.Close analysis of the XRD powder pattern reveals a broad background hump between 35°and 39°2θ (d-spacing between 2.72 and 2.81 Å) and indicates the presence of amorphous hydroxyl apatite.The presence of apatite in a calculus is indicative of hyperparathyroidism. l,2

Discussion
The anomalous intensity patterns are immediately obvious and can arise from several sources.Examples are: orientation effects from abnormal crystal shape due to the mode of crystal growth; 10 particle size effects; degree of pressure during grinding of the sample; loss of water from the crystal structure; isomorphous replacement of some magnesium by calcium, or ammonium by potassium.Our results show that there is no direct correlation between calcium content and the magnitude or pattern of anomalies in the X-ray powder patterns of the struvite in these stones.Where calcium is detected by EDAX or XRF, it is usually associated with solid, amorphous, hydroxyl apatite.
The results show that there are two types of complementary information obtainable.X-Ray powder diffraction immediately identifies the calculus as a 'struvite infection' stone, and thus the patient can be treated appropriately.However, identifying the presence of calcium and/or potassium by EDAX can give an important indication of the underlying problem.For example, calcium, present as amorphous hydroxyl apatite, may indicate a malfunction of the dog's parathyroid gland.If a dog presents a bladder calculus, it may be of value to routinely check the urine for pH as well as for ammonia, which may indicate infection.An excessively high result for potassium may be an indicator for the underlying cause of calculus formation.

Conclusions
We offer the above experiences as a salutary example to other researchers who may be using the X-ray powder diffraction technique.Do not be misled (as we were initially) by 'missing lines' and 'wrong intensities'.These anomalies are not uncommon, and their identification may provide valuable hints of minor but important complexities associated with the condition that generated the stone.Should any readers wish to have urinary calculi analysed by XRD (canine, feline or human), we would be pleased to have them as contributors to this continuing research project.(values of 2θ) are the same for the important lines, but the relative intensities vary greatly.In A the peak at 19°is far higher than the pair at 24°2θ.In E the situation is reversed; the pair at 24°is the strongest.These anomalous intensities can make identifying the calculus as struvite difficult if only the three or four strongest lines are used as the criterion in searching the Powder Diffraction File.Using the seven strongest lines, and ignoring the relative intensities, normally will identify the species as struvite.Neither scan has obvious high backgrounds, nor are there the broad peaks due to poorly crystalline hydroxyl apatite (2.81-2.72Å; 37-38°2θ).Six additional scans of this kind are reproduced in Fig.A online.Table 1.A comparison of the X-ray powder diffraction peaks of synthetic struvite (ICDD data file 15-762) with those struvite calculi recovered from 21 pet dogs.

Fig. 1 .
Fig. 1.Two examples of observed X-ray powder diffraction patterns of struvite calculi.In all cases, the d-spacings (values of 2θ) are the same for the important lines, but the relative intensities vary greatly.In A the peak at 19°is far higher than the pair at 24°2θ.In E the situation is reversed; the pair at 24°is the strongest.These anomalous intensities can make identifying the calculus as struvite difficult if only the three or four strongest lines are used as the criterion in searching the Powder Diffraction File.Using the seven strongest lines, and ignoring the relative intensities, normally will identify the species as struvite.Neither scan has obvious high backgrounds, nor are there the broad peaks due to poorly crystalline hydroxyl apatite (2.81-2.72Å; 37-38°2θ).Six additional scans of this kind are reproduced in Fig.A online.

Table 2 .
Comparison of the unit cell dimensions and X-ray powder diffraction patterns for struvite (NH 4 MgPO 4 @6H 2 O) and the potassium analogue (KMgPO 4 @6H 2 O) in which the K + has replaced the NH 4 + ammonium ion.Data from JCPDS-ICDD (1993) database, struvite data file 15-762, potassium analogue data file 35-812.Note the hkl indices are those given in the Powder File and should be transposed to read hlk for the published cell in 6 and 7.