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Urinary calculi in the dog
J.
COMPo
PATH.
1963. VOL. 73.
119
URINARY CALCULI IN THE DOG*
II.
URATE
STONES
AND
PURINE
METABOLISM
By P.
PORTER
Department of Veterinary Preventive JYledicine, Universiry of Lil'erpool INTRODUCTION
In a previous paper by White, Treacher and Porter (1961) data were presented on the incidence and chemical composition of urinary calculi in the dog. The composition of stones from 122 cases collected between 1957 and 1960 was reported and the stones were classified according to their main constituents as phosphate, oxalate, cystine or urate calculi. The purpose of the present paper is to present further data on the composition of urate calculi and some of the factors in urine which are involved in their formation. MATERIALS AND
METHODS
Sources if Calculi and Urines Urinary calculi were obtained from veterinary hospitals and practising veterinary surgeons in England, Scotland and Wales. Urine samples from these calculus cases were occasionally obtained at surgical operation or at a later period on request. Urine samples from normal dogs were obtained from several breeding kennels in England and the samples were preserved with thymol and stored at temperatures less than 5°C. Qualitative Methods Microchemical analysis if calculi. The routine method of White et ai. (1961) was used. Infra-red spectrophotometry of calculi. Samples were prepared in the form of a paste by mulling with Nujolt in an agate mortar, and analysed using a Perkins Elmer "Infra cord" self-recording spectrophotometer. The spectra were compared with those of purified uric acid, ammonium urate and monosodium urate. The method of preparation of these purified samples will be described in a later paper. Paper chromatography of calculi and urines. A portion of crushed stone was extracted with lithium carbonate and approximately 50 fLl of the solution was spotted onto Whatman NO.4 filter paper and dried under a hair drier to give a compact spot. Urines were electrolytically desalted and then applied to the paper in the same way as the calculi. The chromatogram was developed by allowing a run of approximately 30 cm. with a solvent mixture of n-butanol:diethylene glycol:water (5:r:r) as detailed by Vischer and Chargaff (1948). The purine was identified alongside a standard or its by Rf value, using a method of detection described by Dikstein, Bergmann and Chaimovitz (r956) in which the chromatogram is dipped in or sprayed with a solution of 0'25 per cent. mercuric acetate
* The results presented in this paper formed part of a
t
the University of Liverpool. Spectroscopic grade of liquid paraffin.
thesis for the degree of Ph.D. in
120
URATE STONES AND PURINE METABOLISM
in 95 per cent. alcohol and a little glacial acetic acid which prevents the deposition of mercuric oxide from the solution. The mercuric complexes of the purines thus formed will now give a purple coloration when sprayed with diphenyl carbazone (0'05 per cent. in 95 per cent. ethanol). The purple colour was intensified against the background by drying over a hot plate or in an oven at 60°C. for a few minutes. Quantitative Methods Urate. This was determined by the enzymatic method of Praetorius
and Poulson (1953). Allantoin. The colorimetric method of Larson (1932) was used. Total nitrogen. This was determined by the micro-Kjeldahl method. Ammonia nitrogen. The urine was treated with an equal volume of saturated potassium carbonate and the liberated ammonia was transferred by aeration into an acid of known strength and back-titrated with a standard alkali. Measurement if pH. A Pye Dynacap pH meter with temperature compensation and a full scale deflection of two pH units was used. RESULTS
Calculi In the series of stones from 250 cases received between the years 1957 and 1962 there were 17 cases of urate calculi, in 2 of which the stones recurred. Urate stones thus represent only a small proportion (7.6 per cent.) of the total calculi and are found mainly in the Dalmatian though they do occasionally appear in other breeds. Table 1 shows all the data relating to these 17 cases, including the chemical composition of the stones. Twelve were in Dalmatians and five in other breeds. The stones from four cases had been used up before this work was started in 1959 and so only the results yielded by the incomplete analyses provided by the method of White (1944) are available. The percentage of urate as determined by the uricase enzymatic method, using a weighed amount of stone in a known volume of solution, is expressed in terms of uric acid. The value expressed as ammonium urate is 10 per cent. higher and it is this value which appears in Table I. lrifra-red Spectra The regions of the infra-red spectrum which are most valuable for the qualitative analyses of urates are 2· 5 to 3' 5 fL' where absorption is due to single bonds such as O-H and N-H, 5 to 6'5 fL where absorption is due to double bonds such as C=O and C=C, and above 6'5 fL which is the "finger print" region where there are complicated absorptions due to coupling. Salts do not normally give very satisfactory infra-red spectra, but in the case of urates the spectra proved to be extremely useful for indicating the composition of the calculi. Spectra of purified samples of uric acid, ammonium urate and sodium urate (Fig. I) were prepared in order to act as templates for the analyses of urate calculi.
P. PORTER TABLE
121
I
URATE CALCULUS CASES AND ANALYSIS OF CALCULI
Breed
Age (yr.)
Constituents
Sex
Site
Mqjor
Minor
Ammonium urate (79.6%)
Phosphate oxalate
I
Lakeland Terrier
6
3
Urethral
2
Dalmatian
5
3
Urethral
Urate
3
Spaniel
II
3
Urethral
Ammonium urate (86·5%)
4
Dalmatian
3
3
Urethral
Urate
5
3
Urethral
Urate
2
3
Urethral
Urate Ammonium Urate (82·5%)
Phosphate oxalate
Phosphate oxalate -
5
Dalmatian
6
Dalmatian
7a
Wire Haired Fox Terrier
8
3
Urethral
7b
Wire Haired Fox Terrier (recurrence)
8
3
Urethral
Calcium oxalate
Ammonium urate (26·4%)
8
Spaniel
6
3
Vesical
Ammonium urate (90.75%)
Phosphate
9
Dalmatian
7
3
Urethral
Ammonium urate (88·6%)
Phosphate
10
Boxer
I
3
Urethral
Ammonium urate (7 0 %)
Phosphate
lIa
Dalmatian
7
3
Urethral and vesical
Ammonium urate (39%) Calcium phosphate Triple phosphate
Oxalate
lIb
Dalmatian (recurrence)
7
3
Urethral and vesical
Ammonium urate (59·4%) Calcium phosphate Triple phosphate
Oxalate
12
Dalmatian
It
3
Urethral
Ammonium urate (insufficient)
Phosphate
I
Oxalate
-
I!.Z2
URATE STONES AND PURINE METABOLISM
TABLE I-Continued URATE CALCULUS CASES AND ANALYSIS OF CALCULI
Breed
--
Age (yr.)
Sex
Site
Constituents Major
l.Jinor
13
Dalmatian
lOt
<1
Urethral and vesical
Ammonium urate (88·9%)
Phosphate
14
Dalmatian
6
<1
Renal
Ammonium urate (5 1.3%)
Phosphate
15
Dalmatian
I
<1
Renal, vesical & urethral
Ammonium urate (89.8%)
Phosphate
16
Dalmatian
3
<1
Urethral
Ammonium urate (81·8%)
Phosphate
17
Dalmatian
3
<1
Urethral
Ammonium urate (89·3%)
Phosphate
U ric acid shows the characteristic absorption of the carbonyl group at 5·9 fL. This slight shift to longer wavelength is indicative of co~ugation which is due to the nitrogen lone pair electrons in the molecule. There is a complicated "finger print" region which is characteristic of the molecule. The introduction of the ammonium ion into the molecule produces hydrogen bonding which is shown in ammonium urate by a wide absorption peak at 3'4 fL and a broadening of the carbonyl group absorption peak at 5'9 fL. The "finger print" region is slightly changed from that of uric acid, including the loss of a small absorption peak at 9'7 fL. The spectrum for sodium urate shows considerable change from uric acid and ammonium urate, the characteristic features being absorption due to a hydroxyl group at 2·8 fL' a carbonyl group at 5'75 fL and conjugation of the carbonyl group at 6.05 fL. There is also considerable change in the "finger print" region and a strong indication of aromaticity within the molecule as shown by absorption at wavelengths greater than 13 fL. The infra-red spectra for the urate calculi were always typical of ammonium urate. Two spectra are shown in Fig. 2, one for a urate stone from a Dalmatian and the other for a urate stone found in a Spaniel. They are compared in the same figure with a spectrum of ammonium urate.
Paper Chromatography The use of paper chromatography gives a means of specific identification of the purine components of urine and calculi. As an
12 3
P. PORTER
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13
Infra-red spectra of spectroscopically pure samples. Upper, uric acid; middle, ammonium urate; lower, sodium urate.
c
14
15
124
URATE STONES AND PURINE METABOLISM
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Infra-red spectra of urate calculi compared with ammonium urate. Upper, ammonium urate; middle, urate stone from a Dalmatian (urethral and vesical); lower, urate stone from a Spaniel (urethral).
12 5
P. PORTER
analytical technique for calculi it has considerably greater sensitivity than the murexide test and traces of urate can be indicated where the murexide test fails. Using the solvent system described, the Rf value for uric acid is 0·12 and the value for xanthine is 0·27. Xanthine calculi have been reported in man by Dent and Philpot (1954). There was no evidence of xanthine in the urate calculi from dogs though it was often detected in paper chromatograms of urine.
Urines The analyses of urine samples from normal and calculus-forming dogs are presented in Tables 2, 3,4 and 5. The urine samples for 17 normal Dalmatians were obtained from 6 different breeding kennels in England and covered a wide range of age and strains of the breed. The uricolytic index, the values for which are given in the tables, is an expression derived by Hunter, Givens and Guion (1914) to indicate the extent of the oxidation of uric acid to allantoin. It can be expressed as follows:. .. ( Allantoin N 2 ) Uncolytlc mdex = U· All antOIn . N 2 X 100 . nc aCl·d N 2 (expressed as a percentage). The enzymatic uricase determination using the characteristic ultra-violet absorption at ,\ max 292 mfL is absolutely specific for uric acid. The colorimetric arseno-phosphotungstic acid reaction used in all other work up to the present for the determination of uric acid in dog urine has been demonstrated to give a reaction with an endogenous chromogen in biological fluids (Wolfson, Huddleston and Levine, 1947). Total nitrogen was measured in order to obtain an indication of urine concentration, and the ammonium ion concentration and pH were measured because they were considered to be important factors in urate calculus formation. Table 6 shows the analyses of a series of urines from a Dalmatian after operation for urinary calculi. This animal (Case I I, Table I), had a recurrence within 4 months. Tables 2, 3 and 4 show that Dalmatians in general excrete a considerably greater amount of urate than other breeds. Furthermore, the uricolytic index for the Dalmatian is 36.3 ± 10·9, whereas for other breeds the figure is 92.4 ± 6.02, which emphasises the considerable difference in urate excretion between the Dalmatian and other breeds. In these tables the concentration of urate has also been presented as urate nitrogen expressed as a percentage of total nitrogen, since this takes into account the variation in urinary concentration from time to time and thus permits a reasonable comparison of the figures for individual urines. The percentage of urate nitrogen for Dalmatians (Table 4) lies within the range 0.87 to 3.28, the range of uric acid concentrations being 5 I to 2 I 2·5 mg per cent. There are, however, three dogs of other breeds, with urinary analyses falling within the range found for the Dalmatian, which might be considered to be "high urate excretors". They are
+
~
~
Miniature Dachshund
Scottie
Dachshund
Boxer
Retriever
4
5
6
7
8
~
~
Sheepdog
3
-
-
Sex
2
Terrier
Brsed
Whippet
I
--
;
2!
4
5
3
II
I
14
-
Age (yr,)
.
TABLE 2
5' 6 4 0 24'8
5,87 5,86 7,82 =
89'3
100
Uric acid X 0'33
194
110
I
96
132,6
2620
13 17
1330
1739
9 1 '5
145'5
27 20
4 200
1610
Total nitrogen (mg%)
39 20
78 '9
95'5
96 '6
Uricolytic index
95'8
28 9
23 8 '4
281,6
II6'8
Allantoin (mg%)
-----
* Uric acid N2
18'25
13'15
6,64 6'92
68'7
14'35
4'4
Uric acid (mg%)
6,84
6,69
6'26
pH
I
URINARY ANALYSIS-NORMAL NON-DALMATIANS
13'5
61'7
33'7
39'3
4 1'14
123'5
60'1
47'1
Ammonia (mg%)
I
I
X 100
0'32
0
0'12
0'35
0'11
0,84
0'11
0'09
Total N2
* Uric acid N2
~
en
t:
o
t:>:I
~ t>1 >-l ;I>
t>1
Z
~
'tI
C
Ij
Z
;I>
t>1 en
Z
o
en >-l
~
~
c
Ol
~
TABLE 3
6
7
mths
2
5
6
6
6
if
if
6
if
6
Pekingese
Bull Terrier
Bull Terrier
Boxer
Corgi
Dachshund
King Charles Spaniel
Labrador
I
2a
2b
3
4
5
6
7
II
6
6
4
(yr")
Sex
Breed
Age
I
6"0
7"7
6"02
6"3 6
6"9 2
6"11
6"06
5'94
pH
>I<
I
29 2 "5
I
3682
96 "3 96 "7
3428
87"7
126 20 7"6
2000
94"8
3 116
II83
1286
82"2
1570
9 1"9
2400
98 "7 86"4
Total nitrogen (mg%)
Uricolytic index
169"4
155"6
2 17"4
299"4
622
Allantoin (mg%)
Uric acid NI = Uric acid X 0"33
9"9
8"1
18"8
9"4
35"1
20"4
50 "2
8"5 2
Uric acid (mg%)
I
I
II I
17°"5
261"5
125"2
346
7 1"05
98 "2
161"8
3 25"3
Ammonia (mg%)
URINARY ANALYSIS-NON-DALMATIANS WITH CALCULI OTHER THAN URATE
I
>I<
0"09
0"08
0"3 1
O"II
0"99
0"42
1"06
0"12
Total X 2
Uric acid X 2 X 100
"l
'"
~
~
o
~ 'C
TABLE 4
1I4
6 088
6 °48
6
4
2
if
if
<3
7
8
9
10
if
if
16
17
3 mthso
If
• Uric acid N. = Uric acid x 0033
39° 8
39°2
11.18
121
7° 2
<3
15
115
17°5
39° 1
73
106 °7
5°72
31
<3
14
185° 2
1621 27° 1
37°3
154°4
5"79
2
if
13
6 01
2757 54°9
17608
51
6 °46
6 mthso
<3
12
21I04
262
883
39°3
31
65"6
5° 6
II
if
II
7° 02
201 °4
143 8
36 °5
35°4
212 °5
7°3 6
6
if
II
14°5
280 5
4006
136 °4
33 6 7
2216
~
240
24
5"95
108 3
3° 28
2°37
2°2
1°87
~ 1°93 48 °5
;;:
t"' ~
o
1°48 82 °5
t'l
t'l
Z
'tl
@
t:)
~
o ~ CIJ
~
c
;;:
1°49
X 100
....
00
K)
2°01
61 02
2 11 9
18 08
20-6
94°55
5"77
4
1°7 2
126 06
3260
43°7
12 3° 2
168 06
1°39
9806
6 °5 2
2210
26
3006
1°57
66 °3
92 °4
24 19
33° 1
53
40
1°7 6
75"7
if
6
85° 8
243 2
u18°5
600 9
2004
144°5
33°4
19°3
7
<3
5
541 9
1I6°5
6 °3 6
4
<3
4
1°7 1
133°5
3073
33°7
75°5
157°5
5°99
5
<3
3
0°96
38 °9
3835
41
89° 64
139
7° 1
4
if
2
0 °87
60 °13
3830
2 1°5
25°3 8
100
6 °7 2
9
if
I
Total N.
pH
Sex
* Uric acid N,
Ammonia (mg%)
Total nitrogen (mg%)
Uricolytic index
Allantoin (mg%)
Age (yro)
Uric acid (mg%)
URINARY ANALYSIS-NORMAL DALMATIANS
201'3 120'9
8,69
6,64
6
3
6
6
4
5
- - -
98 '7
5,85
lOi
6
3
- - - - - - _ .. _-_._._--
67'2
136 '4
12,85
192'5
198 4
1553
4 1'1 37'1
777'4
3055
1399
Total nitrogen (mg%)
12'1
63
44
Uricolytic index
4'42 2'03
33 6
4'23
1'3 1
1'59
Total N2
'" Uric acid N2 X 100
595
96
62'4
13 1
---~-
Ammonia (mg%)
Urine (4) was collected post mortem; this dog died as a result of renal calculus, The other urines were taken some time after the operation, '" Uric acid N2 = Uric acid X 0'33,
120
6'12
21 mths,
6
2
48 '63
6'15
7
66,6
6
I
pH
Sex
Allantoin (mg%)
Uric acid (mg%)
Age (yr,)
--
TABLE 5 URINARY ANALYSIS-DALMATIANS WITH CALCULI
...
~
~
~
>0
o
:0
URATE STONES AND PURINE METABOLISM TABLE 6 ANALYSIS OF A SERIES OF URINES AFTER OPERATIONS ON A MALE DALMATIAN
Table
Time
of collection
I,
No.
I I
and Table 5, No.
I
Ammonia (mg%)
Uric acid (mg%)
8'2
3 16
41.1
8'2
3 16
5°·5
18 7
81·1
pH
1St Operation
10/12/59
6.0 p.m.
11/12/59
12.15 p.m.
6'25
11/12/59
4.15 p.m.
6'75
93'5
75·5
12/12/59
10.30 p.m.
6'25
15°·8
68'7
6'15
13°'9
66·6
8'3
299
4°·8
15/12/59 2nd Operation
3/3/60
(a) the Sheepdog (Table 2 NO.3), (b) the Bull Terrier (Table 3 No. 2a) and (c) the Boxer (Table 3 NO.3), with percentage urate nitrogen values of 0.84, I ·06 and 0·99 respectively. The excretion of uric acid has been shown to be affected by a number of dietary factors and so the amount in the urine need not be a function of the urinary concentration. Hence the three dogs so far discussed, whilst having urinary urate levels which fall within the Dalmatian range, show a very different picture when the total excretion of purine end-products is taken into account. Fig. 3 shows that the urate level for the Dalmatian is strongly influenced by the total concentration of purine end-products in that animals having a low urate concentration also have a low excretion of purine end-products as a whole. The main feature differentiating the Sheepdog, Bull Terrier and Boxer, discussed above, from the Dalmatians of comparable urate excretion is that these three excrete very much higher concentrations of purine end-products than the comparable Dalmatians. Thus the Dalmatians which excrete the same level of purine end-products excrete considerably greater levels of urate. Hence on this basis these three animals cannot in fact be classed as "high urate excretors". There is also considerable evidence, presented here, that a high level of urate excretion is not the main criterion in the formation of urate calculi. In the first place there is the fact that urate calculi have been found in other breeds, whereas "high urate excretion" appears to be confined to the Dalmatian. Secondly, when a number of urines from calculus-forming Dalmatians were analysed (Tables
P. PORTER
Fig. 3.
'00 180 160
1<0 120
.'
100
.'
"
80
'0
0:
o
100
'00
'00 Ufol.:'
+
Allol\(oill
",,/.
x DormotiollS
o
Otlu~r ef((cI~
Excretion of end-products of purine metabolism in the dog.
5 and 6) none showed urate concentrations exceeding the range found for normal Dalmatians, though in two cases (Table 5, Nos. 3 and 4) the percentage urate nitrogen values were higher. The latter urine, which also has a high urate concentration, was taken post mortem, and the animal was found to have renal calculi (Case 14, Table I). In this case a high uric acid excretion would probably have played an important part in calculus formation at this site by promoting rapid growth of the stone. DISCUSSION
Urate calculi are usually small and spherical and when broken are seen to be made up of concentric laminations. All the cases were in male dogs. It is likely that the bitch would expel a urate stone without showing symptoms, whereas the narrow groove of the os penis of the male dog presents considerable hindrance to the passage of even the smallest stones. The composition of the calculi as determined by the infra-red spectrum was always ammonium urate and never, as is almost universal in human cases, uric acid. Prien and Fronde! (1949), in their identification of calculi by X-ray crystallographic and polarographic techniques, reported an ammonium urate calculus from a Bulldog whereas all their human cases had uric acid stones. It would appear, therefore, that the ammonium ion may playa part in urate calculus formation in the dog. This is also in keeping with theoretical reasoning, since the solubility results of Gudzent (1908)
URATE STONES AND PURINE METABOLISM
showed that ammonium urate is the least soluble salt of uric acid. A further point of interest is that most urate calculi contain some phosphate. When a series of calculi were analysed using paper chromatography all the phosphate stones showed traces of urate whereas none was present in oxalate or cystine stones. White (1944) reported similar findings in 26 phosphate calculi, using the murexide test. It is thus possible that a connection exists between the deposition of phosphates and urates from urine, and that the conditions influencing the precipitation of the two are similar, or that the precipitation of one immediately involves the precipitation of the other. Benedict ( 1916) made the surprising discovery that the Dalmatian was unique among dog breeds and exceptional to the uricolytic index classification of Hunter et al. (1914) in that it excreted as much uric acid per day as a man. Calculation from Benedict's results for uric acid and allantoin show the Dalmatian to have a uricolytic index of approximately 36 which is about midway between the values for man and dogs in general. Wells (1918) demonstrated that the liver and spleen of the Dalmatian possessed uricolytic activity, hence the high uric acid excretion was not due to the absence of the enzyme uricase. Klemperer, Trimble and Hastings (1938) demonstrated that the uricolytic activity ofliver slices from the Dalmatian was substantially the same as in other breeds. Friedman and Byers (1948) established that the main cause of the excretion of high levels of uric acid in the Dalmatian is a renal anomaly whereby uric acid is excreted in the glomerular filtrate and fails to be reabsorbed in the tubules. Trimble and Keeler (1938), using a family of Dalmatians, crossed them with a collie dog and a collie bitch. The progeny were in two groups, "low uric acid excretors" and "high uric acid excretors", sharply separated and with no intermediate grade. Thus the data available in the literature suggest that the Dalmatian alone among breeds is likely to develop urate calculi, but there have in fact been a number of reports of urate stones in other breeds. White (1944) reported urate stones in an Airedale, a Bulldog and a Welsh Corgi, while Prien and Frondel (1949) mentioned an ammonium urate calculus in a Bulldog. Brodey (1955), in a survey of 52 clinical cases of canine urolithiasis, reported IO uric acid calculi, 4 in Dalmatians, 2 in Dachshunds, 2 in Irish Terriers, 1 in a mongrel and 1 in a Chihuahua, but the method of detection makes it possible that cystine and not uric acid may sometimes have been the constituent. White et al. (1961) reported urate calculi from a Boxer, 2 Spaniels, a Fox Terrier and a Lakeland Terrier, and these cases are included in this paper. The ability to excrete high levels of uric acid might not, therefore, be entirely confined to the Dalmatian and, contrary to the observations of Trimble and Keeler (1938), the Dalmatian might merely occupy a position at the head of the list of breeds and excrete the highest levels of urate.
P. PORTER
133
The present investigations show that other breeds may excrete concentrations of urate within the lower range observed for the Dalmatian, but only by simultaneously excreting high concentrations of allantoin. Keeler (1940) observed that the Dalmatian excreted eleven times as much uric acid as other breeds and suggested that this would be the predisposing factor contributing to the high incidence of renal stones. The results reported in this paper show that in urate calculus disease the site at which the stones were found in our series was mainly the urethra of the male. Renal urate stones were rare (2 of 17 cases) and it is in fact our experience that renal calculus formation in the dog involving any of the four chemical types of stone is relatively rare (White et al. 1961). We have not, however, conducted a post-mortem survey, but have depended mainly on specimens sent to us from practitioners. The results presented in this paper indicate that urate calculus cases do not excrete any greater quantities of urate than the normal Dalmatian and that high urate excretion is not the main requirement for urate calculus disease. In fact one Dalmatian, which formed calculi composed of a mixture of urate and phosphate, excreted urate at only a very low concentration. A well esatablished theory of medical literature, based entirely on solubility data and a number of observations in man, is that urate or uric acid calculi are caused by low urinary pH. Atsmon, Frank, Lazebnik, Kochwa and de Vries (1960) studied 58 human patients with urate calculus disease and observed that the morning urine was unusually acidic (pH 5). Henneman, Wallack and Dempsey (1958) suggested that the responsibility for urate calculus formation lay in a metabolic defect in which insufficient ammonia production in the renal tubules created a low urinary pH. In the cases observed in the Dalmatian, however, none has shown an abnormally low pH and in urines taken at operation the pH has been very high, and indeed abnormally high ammonia levels have been observed. This increase is possibly due to inflammation and infection. Infections, with the presence of urea-splitting organisms, have been regarded as important factors in stone formation since the organisms by producing ammonia increase the ~lkalinity of the urine, thus predisposing to the precipitation of phosphates. Whilst uric acid becomes more soluble with increasing pH, ammonium urate under such conditions would become less soluble due to the increased ammonium ion concentration: hence there would be an increased tendency for the precipitation of ammonium urate which is in fact the least soluble salt of uric acid. This would possibly explain the observation that urates and phosphates are frequently mixed in calculi. The urinary pH rapidly returns to normal after removal of the calculi. Dietary factors are considered to be important in the production of calculi. Vitamin A deficiency has been cited as a factor in stone formation mainly because of the experimental production of calculi
134
URATE STONES AND PURINE METABOLISM
in rats by Higgins (1951) with a vitamin A deficient diets Dalmatians on a diet deficient in Vitamin A also formed uric acid stones. It has been considered that a necessary measure in prevention of urate calculi is the feeding of a low purine diet. Brewer (1954), in treating a case of uric acid stones in a Dalmatian, suggested such foods low in purines as cheese, eggs, milk, cereals, fruit and vegetables and advocated that fats should be avoided, but Young, Conway and Crandall (1938), in metabolic experiments using synthetic diets, found that though the uricolytic index varied with the diet and the individual Dalmatian, there was no conclusive evidence for a particular dietary precursor in the synthesis of uric acid. Guanine, xanthine, uric acid and nucleic acids administered in capsule form increased the output of allantoin and urea but not of uric acid, and the excretion of uric acid was so constant that they suggested it was an endogenous metabolite. On the basis of these experiments there would thus appear to be little value in controlling the purine intake. The control of urinary pH by feeding alkaline salts such as potassium citrate could be of value, but the indications are that the problem of urate stones in the dog is different from that in man. The effect of the ammonium ion concentration, in the first instance, would appear to be more important than the pH in influencing urate calculus formation in the dog, and though an increase of pH would certainly increase the solubility of uric acid it would have no effect on the solubility of ammonium urate which is a poorly dissociated salt. Experiments to evaluate the efficiency of alkaline salts in the control of urinary ammonium ion concentration will be reported later. Alkalinisation of the urine may result in other complications, however, since it involves the danger of inducing the precipitation of phosphates. These observations emphasise the need for further study of the factors predisposing to the precipitation of urates and this forms the subject of further papers. CONCLUSIONS
Analysis of urate calculi from 17 cases in dogs using a microchemical technique, paper chromatography, ultra-violet and infrared spectrophotometry has shown that the main constituent is ammonium urate, and that some phosphate is usually present as well. Urate stones were found mainly in the Dalmatian (12 cases) but also in other breeds (5 cases). The determination of uric acid and allantoin in urine samples d h h . I . . d (AllantoinN 2 ) . d' lcate t att euncoytlcIll ex U· . N 2 XIOO nc A Cl'dN 2 +AII antOln of the Dalmatian was 36'3 ± 10'9 and of other breeds 92'4 ± 6'02. Although individuals in other breeds may excrete urate within the lower limits found for the Dalmatian they only do so by simultaneously excreting high concentrations of allantoiD., i.e. they have a high uricolytic index. III
P. PORTER
135
The concentration of uric acid in the urine of calculus-forming Dalmatians was no greater than in normal dogs of this breed and the level of uric acid in the urine does not appear to be the important factor in urate calculus formation. In contrast to the position with uric acid calculi in man, the urine in Dalmatians with urate calculi was not unusually acidic. The ammonium ion concentration, however, appears to be an important factor in urate calculus formation in the dog. ACKNOWLEDGMENTS
I am indebted to all the veterinary surgeons and dog breeders who supplied calculi, urines and details of the animals concerned, to Professor E. G. White for his constant interest and advice, and the Wellcome Trust for a grant which covered the cost of much of the work. REFERENCES
Atsmon, A., Frank, M., Lazebnik, j., Kochwa, S., and de Vries, A. (1960).]. Uro!.,84, 167. Benedict, S. R. (1916).]. Lab. clin. Med., 2 I. Brewer, N. R. (1954), N. Amer. vet., 35, 372. Brodey, R. S. (1955).]. Amer. vet. 'II1ed. Ass., 126, I. Dent, C. E., and Philpot, G. R. (1954). Lancet, 266, 182. Dikstein, S., Bergmann, F., and Chaimovitz, M. (1956). ]. biol. Chem., 221,239· Friedman, M., and Byers, S. O. (1948). Ibid., 175, 727. Gudzent, F. (1908). Z. physiol. Chem., 56, 150. Henneman, P. H., Wallack, S., and Dempsey, R. F. (1958). ]. clin. Invest., 37, 901. Higgins, C. C. (1951). ]. Amer. vet. med. Ass., lI8, 81. Hunter, A., Givens, M. H., and Guion, A. N. (1914), ]. biol. Chem., 18, 387. Keeler, C. E. (1940).]. Amer. vet. med. Ass., 96,507. Klemperer, F. W., Trimble, H. C., and Hastings, A. B. (1938). ]. biol. Chem., 125, 445. Larson, H. W. (1932). Ibid., 94, 727. Praetorius, E. A., and Poulson, H. E. (1953)' Scand. ]. clin. Lab. Invest., 5, 273. Prien, E. L., and Frondel, C. (1949).]. Urol., 57,949. Trimble, H. C., and Keeler, C. E. (1938).]. Hered., 30,281. Vischer, E., and Chargaff, E. (1948).]. bio!. Chem., 176, 703. Wells, H. G. (1918). Ibid., 35,221. White, E. G. (1944),], compo Path., 54, 16. White, E. G., Treacher, R. j., and Porter P. (1961). Ibid., 71.,201. Wolfson, W. Q., Huddleston, B., and Levine, R. (1947). ]. clin. Invest. 26, 995. Young, E. G., Conway, C. F., and Crandall, W. A. (1938). Biochem. ]., 32, 1138. [Received for publication, October 22nd, 1962]
COMPo
PATH.
1963. VOL. 73.
119
URINARY CALCULI IN THE DOG*
II.
URATE
STONES
AND
PURINE
METABOLISM
By P.
PORTER
Department of Veterinary Preventive JYledicine, Universiry of Lil'erpool INTRODUCTION
In a previous paper by White, Treacher and Porter (1961) data were presented on the incidence and chemical composition of urinary calculi in the dog. The composition of stones from 122 cases collected between 1957 and 1960 was reported and the stones were classified according to their main constituents as phosphate, oxalate, cystine or urate calculi. The purpose of the present paper is to present further data on the composition of urate calculi and some of the factors in urine which are involved in their formation. MATERIALS AND
METHODS
Sources if Calculi and Urines Urinary calculi were obtained from veterinary hospitals and practising veterinary surgeons in England, Scotland and Wales. Urine samples from these calculus cases were occasionally obtained at surgical operation or at a later period on request. Urine samples from normal dogs were obtained from several breeding kennels in England and the samples were preserved with thymol and stored at temperatures less than 5°C. Qualitative Methods Microchemical analysis if calculi. The routine method of White et ai. (1961) was used. Infra-red spectrophotometry of calculi. Samples were prepared in the form of a paste by mulling with Nujolt in an agate mortar, and analysed using a Perkins Elmer "Infra cord" self-recording spectrophotometer. The spectra were compared with those of purified uric acid, ammonium urate and monosodium urate. The method of preparation of these purified samples will be described in a later paper. Paper chromatography of calculi and urines. A portion of crushed stone was extracted with lithium carbonate and approximately 50 fLl of the solution was spotted onto Whatman NO.4 filter paper and dried under a hair drier to give a compact spot. Urines were electrolytically desalted and then applied to the paper in the same way as the calculi. The chromatogram was developed by allowing a run of approximately 30 cm. with a solvent mixture of n-butanol:diethylene glycol:water (5:r:r) as detailed by Vischer and Chargaff (1948). The purine was identified alongside a standard or its by Rf value, using a method of detection described by Dikstein, Bergmann and Chaimovitz (r956) in which the chromatogram is dipped in or sprayed with a solution of 0'25 per cent. mercuric acetate
* The results presented in this paper formed part of a
t
the University of Liverpool. Spectroscopic grade of liquid paraffin.
thesis for the degree of Ph.D. in
120
URATE STONES AND PURINE METABOLISM
in 95 per cent. alcohol and a little glacial acetic acid which prevents the deposition of mercuric oxide from the solution. The mercuric complexes of the purines thus formed will now give a purple coloration when sprayed with diphenyl carbazone (0'05 per cent. in 95 per cent. ethanol). The purple colour was intensified against the background by drying over a hot plate or in an oven at 60°C. for a few minutes. Quantitative Methods Urate. This was determined by the enzymatic method of Praetorius
and Poulson (1953). Allantoin. The colorimetric method of Larson (1932) was used. Total nitrogen. This was determined by the micro-Kjeldahl method. Ammonia nitrogen. The urine was treated with an equal volume of saturated potassium carbonate and the liberated ammonia was transferred by aeration into an acid of known strength and back-titrated with a standard alkali. Measurement if pH. A Pye Dynacap pH meter with temperature compensation and a full scale deflection of two pH units was used. RESULTS
Calculi In the series of stones from 250 cases received between the years 1957 and 1962 there were 17 cases of urate calculi, in 2 of which the stones recurred. Urate stones thus represent only a small proportion (7.6 per cent.) of the total calculi and are found mainly in the Dalmatian though they do occasionally appear in other breeds. Table 1 shows all the data relating to these 17 cases, including the chemical composition of the stones. Twelve were in Dalmatians and five in other breeds. The stones from four cases had been used up before this work was started in 1959 and so only the results yielded by the incomplete analyses provided by the method of White (1944) are available. The percentage of urate as determined by the uricase enzymatic method, using a weighed amount of stone in a known volume of solution, is expressed in terms of uric acid. The value expressed as ammonium urate is 10 per cent. higher and it is this value which appears in Table I. lrifra-red Spectra The regions of the infra-red spectrum which are most valuable for the qualitative analyses of urates are 2· 5 to 3' 5 fL' where absorption is due to single bonds such as O-H and N-H, 5 to 6'5 fL where absorption is due to double bonds such as C=O and C=C, and above 6'5 fL which is the "finger print" region where there are complicated absorptions due to coupling. Salts do not normally give very satisfactory infra-red spectra, but in the case of urates the spectra proved to be extremely useful for indicating the composition of the calculi. Spectra of purified samples of uric acid, ammonium urate and sodium urate (Fig. I) were prepared in order to act as templates for the analyses of urate calculi.
P. PORTER TABLE
121
I
URATE CALCULUS CASES AND ANALYSIS OF CALCULI
Breed
Age (yr.)
Constituents
Sex
Site
Mqjor
Minor
Ammonium urate (79.6%)
Phosphate oxalate
I
Lakeland Terrier
6
3
Urethral
2
Dalmatian
5
3
Urethral
Urate
3
Spaniel
II
3
Urethral
Ammonium urate (86·5%)
4
Dalmatian
3
3
Urethral
Urate
5
3
Urethral
Urate
2
3
Urethral
Urate Ammonium Urate (82·5%)
Phosphate oxalate
Phosphate oxalate -
5
Dalmatian
6
Dalmatian
7a
Wire Haired Fox Terrier
8
3
Urethral
7b
Wire Haired Fox Terrier (recurrence)
8
3
Urethral
Calcium oxalate
Ammonium urate (26·4%)
8
Spaniel
6
3
Vesical
Ammonium urate (90.75%)
Phosphate
9
Dalmatian
7
3
Urethral
Ammonium urate (88·6%)
Phosphate
10
Boxer
I
3
Urethral
Ammonium urate (7 0 %)
Phosphate
lIa
Dalmatian
7
3
Urethral and vesical
Ammonium urate (39%) Calcium phosphate Triple phosphate
Oxalate
lIb
Dalmatian (recurrence)
7
3
Urethral and vesical
Ammonium urate (59·4%) Calcium phosphate Triple phosphate
Oxalate
12
Dalmatian
It
3
Urethral
Ammonium urate (insufficient)
Phosphate
I
Oxalate
-
I!.Z2
URATE STONES AND PURINE METABOLISM
TABLE I-Continued URATE CALCULUS CASES AND ANALYSIS OF CALCULI
Breed
--
Age (yr.)
Sex
Site
Constituents Major
l.Jinor
13
Dalmatian
lOt
<1
Urethral and vesical
Ammonium urate (88·9%)
Phosphate
14
Dalmatian
6
<1
Renal
Ammonium urate (5 1.3%)
Phosphate
15
Dalmatian
I
<1
Renal, vesical & urethral
Ammonium urate (89.8%)
Phosphate
16
Dalmatian
3
<1
Urethral
Ammonium urate (81·8%)
Phosphate
17
Dalmatian
3
<1
Urethral
Ammonium urate (89·3%)
Phosphate
U ric acid shows the characteristic absorption of the carbonyl group at 5·9 fL. This slight shift to longer wavelength is indicative of co~ugation which is due to the nitrogen lone pair electrons in the molecule. There is a complicated "finger print" region which is characteristic of the molecule. The introduction of the ammonium ion into the molecule produces hydrogen bonding which is shown in ammonium urate by a wide absorption peak at 3'4 fL and a broadening of the carbonyl group absorption peak at 5'9 fL. The "finger print" region is slightly changed from that of uric acid, including the loss of a small absorption peak at 9'7 fL. The spectrum for sodium urate shows considerable change from uric acid and ammonium urate, the characteristic features being absorption due to a hydroxyl group at 2·8 fL' a carbonyl group at 5'75 fL and conjugation of the carbonyl group at 6.05 fL. There is also considerable change in the "finger print" region and a strong indication of aromaticity within the molecule as shown by absorption at wavelengths greater than 13 fL. The infra-red spectra for the urate calculi were always typical of ammonium urate. Two spectra are shown in Fig. 2, one for a urate stone from a Dalmatian and the other for a urate stone found in a Spaniel. They are compared in the same figure with a spectrum of ammonium urate.
Paper Chromatography The use of paper chromatography gives a means of specific identification of the purine components of urine and calculi. As an
12 3
P. PORTER
Fig. 4000 3000 100
al2
80
CM-I
1500
2000
I.
1000
900
10
11
800
700
\ !~
LU
~ 60
1/
«
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40
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«
~ 20 0
4
3
4000 3000 100
al2
5
6
7
« l-
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1000
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w
I-
WAVELENGTH (MICRONS)
1500
2000
9
8
"j
N
40
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-
..... \I
INFRACORO ••
13
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14
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Z
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~ 20
a
3
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4000 3000 100
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5
6
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WAVELENGTH (MICRONS)
1000
.V
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w
~ 60
Ir-
.....
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SE
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o
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4
5
6
7
8
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WAVELENGTH (MICRONS)
11
I
12
INFRACORD,.',.:'137-t281
13
Infra-red spectra of spectroscopically pure samples. Upper, uric acid; middle, ammonium urate; lower, sodium urate.
c
14
15
124
URATE STONES AND PURINE METABOLISM
Fig. 4000 3000 10 0
2000
2.
CM·'
1500
1-
/II
~8 0
'--
1000
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V
900
800
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V
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, 0-:;;:
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,
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INFRACORO •• '
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1.1
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r--
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3
4
5
6
7
8
9
10
WAVElENGTH (MICRONS)
11
12
13
14
1371281
15
Infra-red spectra of urate calculi compared with ammonium urate. Upper, ammonium urate; middle, urate stone from a Dalmatian (urethral and vesical); lower, urate stone from a Spaniel (urethral).
12 5
P. PORTER
analytical technique for calculi it has considerably greater sensitivity than the murexide test and traces of urate can be indicated where the murexide test fails. Using the solvent system described, the Rf value for uric acid is 0·12 and the value for xanthine is 0·27. Xanthine calculi have been reported in man by Dent and Philpot (1954). There was no evidence of xanthine in the urate calculi from dogs though it was often detected in paper chromatograms of urine.
Urines The analyses of urine samples from normal and calculus-forming dogs are presented in Tables 2, 3,4 and 5. The urine samples for 17 normal Dalmatians were obtained from 6 different breeding kennels in England and covered a wide range of age and strains of the breed. The uricolytic index, the values for which are given in the tables, is an expression derived by Hunter, Givens and Guion (1914) to indicate the extent of the oxidation of uric acid to allantoin. It can be expressed as follows:. .. ( Allantoin N 2 ) Uncolytlc mdex = U· All antOIn . N 2 X 100 . nc aCl·d N 2 (expressed as a percentage). The enzymatic uricase determination using the characteristic ultra-violet absorption at ,\ max 292 mfL is absolutely specific for uric acid. The colorimetric arseno-phosphotungstic acid reaction used in all other work up to the present for the determination of uric acid in dog urine has been demonstrated to give a reaction with an endogenous chromogen in biological fluids (Wolfson, Huddleston and Levine, 1947). Total nitrogen was measured in order to obtain an indication of urine concentration, and the ammonium ion concentration and pH were measured because they were considered to be important factors in urate calculus formation. Table 6 shows the analyses of a series of urines from a Dalmatian after operation for urinary calculi. This animal (Case I I, Table I), had a recurrence within 4 months. Tables 2, 3 and 4 show that Dalmatians in general excrete a considerably greater amount of urate than other breeds. Furthermore, the uricolytic index for the Dalmatian is 36.3 ± 10·9, whereas for other breeds the figure is 92.4 ± 6.02, which emphasises the considerable difference in urate excretion between the Dalmatian and other breeds. In these tables the concentration of urate has also been presented as urate nitrogen expressed as a percentage of total nitrogen, since this takes into account the variation in urinary concentration from time to time and thus permits a reasonable comparison of the figures for individual urines. The percentage of urate nitrogen for Dalmatians (Table 4) lies within the range 0.87 to 3.28, the range of uric acid concentrations being 5 I to 2 I 2·5 mg per cent. There are, however, three dogs of other breeds, with urinary analyses falling within the range found for the Dalmatian, which might be considered to be "high urate excretors". They are
+
~
~
Miniature Dachshund
Scottie
Dachshund
Boxer
Retriever
4
5
6
7
8
~
~
Sheepdog
3
-
-
Sex
2
Terrier
Brsed
Whippet
I
--
;
2!
4
5
3
II
I
14
-
Age (yr,)
.
TABLE 2
5' 6 4 0 24'8
5,87 5,86 7,82 =
89'3
100
Uric acid X 0'33
194
110
I
96
132,6
2620
13 17
1330
1739
9 1 '5
145'5
27 20
4 200
1610
Total nitrogen (mg%)
39 20
78 '9
95'5
96 '6
Uricolytic index
95'8
28 9
23 8 '4
281,6
II6'8
Allantoin (mg%)
-----
* Uric acid N2
18'25
13'15
6,64 6'92
68'7
14'35
4'4
Uric acid (mg%)
6,84
6,69
6'26
pH
I
URINARY ANALYSIS-NORMAL NON-DALMATIANS
13'5
61'7
33'7
39'3
4 1'14
123'5
60'1
47'1
Ammonia (mg%)
I
I
X 100
0'32
0
0'12
0'35
0'11
0,84
0'11
0'09
Total N2
* Uric acid N2
~
en
t:
o
t:>:I
~ t>1 >-l ;I>
t>1
Z
~
'tI
C
Ij
Z
;I>
t>1 en
Z
o
en >-l
~
~
c
Ol
~
TABLE 3
6
7
mths
2
5
6
6
6
if
if
6
if
6
Pekingese
Bull Terrier
Bull Terrier
Boxer
Corgi
Dachshund
King Charles Spaniel
Labrador
I
2a
2b
3
4
5
6
7
II
6
6
4
(yr")
Sex
Breed
Age
I
6"0
7"7
6"02
6"3 6
6"9 2
6"11
6"06
5'94
pH
>I<
I
29 2 "5
I
3682
96 "3 96 "7
3428
87"7
126 20 7"6
2000
94"8
3 116
II83
1286
82"2
1570
9 1"9
2400
98 "7 86"4
Total nitrogen (mg%)
Uricolytic index
169"4
155"6
2 17"4
299"4
622
Allantoin (mg%)
Uric acid NI = Uric acid X 0"33
9"9
8"1
18"8
9"4
35"1
20"4
50 "2
8"5 2
Uric acid (mg%)
I
I
II I
17°"5
261"5
125"2
346
7 1"05
98 "2
161"8
3 25"3
Ammonia (mg%)
URINARY ANALYSIS-NON-DALMATIANS WITH CALCULI OTHER THAN URATE
I
>I<
0"09
0"08
0"3 1
O"II
0"99
0"42
1"06
0"12
Total X 2
Uric acid X 2 X 100
"l
'"
~
~
o
~ 'C
TABLE 4
1I4
6 088
6 °48
6
4
2
if
if
<3
7
8
9
10
if
if
16
17
3 mthso
If
• Uric acid N. = Uric acid x 0033
39° 8
39°2
11.18
121
7° 2
<3
15
115
17°5
39° 1
73
106 °7
5°72
31
<3
14
185° 2
1621 27° 1
37°3
154°4
5"79
2
if
13
6 01
2757 54°9
17608
51
6 °46
6 mthso
<3
12
21I04
262
883
39°3
31
65"6
5° 6
II
if
II
7° 02
201 °4
143 8
36 °5
35°4
212 °5
7°3 6
6
if
II
14°5
280 5
4006
136 °4
33 6 7
2216
~
240
24
5"95
108 3
3° 28
2°37
2°2
1°87
~ 1°93 48 °5
;;:
t"' ~
o
1°48 82 °5
t'l
t'l
Z
'tl
@
t:)
~
o ~ CIJ
~
c
;;:
1°49
X 100
....
00
K)
2°01
61 02
2 11 9
18 08
20-6
94°55
5"77
4
1°7 2
126 06
3260
43°7
12 3° 2
168 06
1°39
9806
6 °5 2
2210
26
3006
1°57
66 °3
92 °4
24 19
33° 1
53
40
1°7 6
75"7
if
6
85° 8
243 2
u18°5
600 9
2004
144°5
33°4
19°3
7
<3
5
541 9
1I6°5
6 °3 6
4
<3
4
1°7 1
133°5
3073
33°7
75°5
157°5
5°99
5
<3
3
0°96
38 °9
3835
41
89° 64
139
7° 1
4
if
2
0 °87
60 °13
3830
2 1°5
25°3 8
100
6 °7 2
9
if
I
Total N.
pH
Sex
* Uric acid N,
Ammonia (mg%)
Total nitrogen (mg%)
Uricolytic index
Allantoin (mg%)
Age (yro)
Uric acid (mg%)
URINARY ANALYSIS-NORMAL DALMATIANS
201'3 120'9
8,69
6,64
6
3
6
6
4
5
- - -
98 '7
5,85
lOi
6
3
- - - - - - _ .. _-_._._--
67'2
136 '4
12,85
192'5
198 4
1553
4 1'1 37'1
777'4
3055
1399
Total nitrogen (mg%)
12'1
63
44
Uricolytic index
4'42 2'03
33 6
4'23
1'3 1
1'59
Total N2
'" Uric acid N2 X 100
595
96
62'4
13 1
---~-
Ammonia (mg%)
Urine (4) was collected post mortem; this dog died as a result of renal calculus, The other urines were taken some time after the operation, '" Uric acid N2 = Uric acid X 0'33,
120
6'12
21 mths,
6
2
48 '63
6'15
7
66,6
6
I
pH
Sex
Allantoin (mg%)
Uric acid (mg%)
Age (yr,)
--
TABLE 5 URINARY ANALYSIS-DALMATIANS WITH CALCULI
...
~
~
~
>0
o
:0
URATE STONES AND PURINE METABOLISM TABLE 6 ANALYSIS OF A SERIES OF URINES AFTER OPERATIONS ON A MALE DALMATIAN
Table
Time
of collection
I,
No.
I I
and Table 5, No.
I
Ammonia (mg%)
Uric acid (mg%)
8'2
3 16
41.1
8'2
3 16
5°·5
18 7
81·1
pH
1St Operation
10/12/59
6.0 p.m.
11/12/59
12.15 p.m.
6'25
11/12/59
4.15 p.m.
6'75
93'5
75·5
12/12/59
10.30 p.m.
6'25
15°·8
68'7
6'15
13°'9
66·6
8'3
299
4°·8
15/12/59 2nd Operation
3/3/60
(a) the Sheepdog (Table 2 NO.3), (b) the Bull Terrier (Table 3 No. 2a) and (c) the Boxer (Table 3 NO.3), with percentage urate nitrogen values of 0.84, I ·06 and 0·99 respectively. The excretion of uric acid has been shown to be affected by a number of dietary factors and so the amount in the urine need not be a function of the urinary concentration. Hence the three dogs so far discussed, whilst having urinary urate levels which fall within the Dalmatian range, show a very different picture when the total excretion of purine end-products is taken into account. Fig. 3 shows that the urate level for the Dalmatian is strongly influenced by the total concentration of purine end-products in that animals having a low urate concentration also have a low excretion of purine end-products as a whole. The main feature differentiating the Sheepdog, Bull Terrier and Boxer, discussed above, from the Dalmatians of comparable urate excretion is that these three excrete very much higher concentrations of purine end-products than the comparable Dalmatians. Thus the Dalmatians which excrete the same level of purine end-products excrete considerably greater levels of urate. Hence on this basis these three animals cannot in fact be classed as "high urate excretors". There is also considerable evidence, presented here, that a high level of urate excretion is not the main criterion in the formation of urate calculi. In the first place there is the fact that urate calculi have been found in other breeds, whereas "high urate excretion" appears to be confined to the Dalmatian. Secondly, when a number of urines from calculus-forming Dalmatians were analysed (Tables
P. PORTER
Fig. 3.
'00 180 160
1<0 120
.'
100
.'
"
80
'0
0:
o
100
'00
'00 Ufol.:'
+
Allol\(oill
",,/.
x DormotiollS
o
Otlu~r ef((cI~
Excretion of end-products of purine metabolism in the dog.
5 and 6) none showed urate concentrations exceeding the range found for normal Dalmatians, though in two cases (Table 5, Nos. 3 and 4) the percentage urate nitrogen values were higher. The latter urine, which also has a high urate concentration, was taken post mortem, and the animal was found to have renal calculi (Case 14, Table I). In this case a high uric acid excretion would probably have played an important part in calculus formation at this site by promoting rapid growth of the stone. DISCUSSION
Urate calculi are usually small and spherical and when broken are seen to be made up of concentric laminations. All the cases were in male dogs. It is likely that the bitch would expel a urate stone without showing symptoms, whereas the narrow groove of the os penis of the male dog presents considerable hindrance to the passage of even the smallest stones. The composition of the calculi as determined by the infra-red spectrum was always ammonium urate and never, as is almost universal in human cases, uric acid. Prien and Fronde! (1949), in their identification of calculi by X-ray crystallographic and polarographic techniques, reported an ammonium urate calculus from a Bulldog whereas all their human cases had uric acid stones. It would appear, therefore, that the ammonium ion may playa part in urate calculus formation in the dog. This is also in keeping with theoretical reasoning, since the solubility results of Gudzent (1908)
URATE STONES AND PURINE METABOLISM
showed that ammonium urate is the least soluble salt of uric acid. A further point of interest is that most urate calculi contain some phosphate. When a series of calculi were analysed using paper chromatography all the phosphate stones showed traces of urate whereas none was present in oxalate or cystine stones. White (1944) reported similar findings in 26 phosphate calculi, using the murexide test. It is thus possible that a connection exists between the deposition of phosphates and urates from urine, and that the conditions influencing the precipitation of the two are similar, or that the precipitation of one immediately involves the precipitation of the other. Benedict ( 1916) made the surprising discovery that the Dalmatian was unique among dog breeds and exceptional to the uricolytic index classification of Hunter et al. (1914) in that it excreted as much uric acid per day as a man. Calculation from Benedict's results for uric acid and allantoin show the Dalmatian to have a uricolytic index of approximately 36 which is about midway between the values for man and dogs in general. Wells (1918) demonstrated that the liver and spleen of the Dalmatian possessed uricolytic activity, hence the high uric acid excretion was not due to the absence of the enzyme uricase. Klemperer, Trimble and Hastings (1938) demonstrated that the uricolytic activity ofliver slices from the Dalmatian was substantially the same as in other breeds. Friedman and Byers (1948) established that the main cause of the excretion of high levels of uric acid in the Dalmatian is a renal anomaly whereby uric acid is excreted in the glomerular filtrate and fails to be reabsorbed in the tubules. Trimble and Keeler (1938), using a family of Dalmatians, crossed them with a collie dog and a collie bitch. The progeny were in two groups, "low uric acid excretors" and "high uric acid excretors", sharply separated and with no intermediate grade. Thus the data available in the literature suggest that the Dalmatian alone among breeds is likely to develop urate calculi, but there have in fact been a number of reports of urate stones in other breeds. White (1944) reported urate stones in an Airedale, a Bulldog and a Welsh Corgi, while Prien and Frondel (1949) mentioned an ammonium urate calculus in a Bulldog. Brodey (1955), in a survey of 52 clinical cases of canine urolithiasis, reported IO uric acid calculi, 4 in Dalmatians, 2 in Dachshunds, 2 in Irish Terriers, 1 in a mongrel and 1 in a Chihuahua, but the method of detection makes it possible that cystine and not uric acid may sometimes have been the constituent. White et al. (1961) reported urate calculi from a Boxer, 2 Spaniels, a Fox Terrier and a Lakeland Terrier, and these cases are included in this paper. The ability to excrete high levels of uric acid might not, therefore, be entirely confined to the Dalmatian and, contrary to the observations of Trimble and Keeler (1938), the Dalmatian might merely occupy a position at the head of the list of breeds and excrete the highest levels of urate.
P. PORTER
133
The present investigations show that other breeds may excrete concentrations of urate within the lower range observed for the Dalmatian, but only by simultaneously excreting high concentrations of allantoin. Keeler (1940) observed that the Dalmatian excreted eleven times as much uric acid as other breeds and suggested that this would be the predisposing factor contributing to the high incidence of renal stones. The results reported in this paper show that in urate calculus disease the site at which the stones were found in our series was mainly the urethra of the male. Renal urate stones were rare (2 of 17 cases) and it is in fact our experience that renal calculus formation in the dog involving any of the four chemical types of stone is relatively rare (White et al. 1961). We have not, however, conducted a post-mortem survey, but have depended mainly on specimens sent to us from practitioners. The results presented in this paper indicate that urate calculus cases do not excrete any greater quantities of urate than the normal Dalmatian and that high urate excretion is not the main requirement for urate calculus disease. In fact one Dalmatian, which formed calculi composed of a mixture of urate and phosphate, excreted urate at only a very low concentration. A well esatablished theory of medical literature, based entirely on solubility data and a number of observations in man, is that urate or uric acid calculi are caused by low urinary pH. Atsmon, Frank, Lazebnik, Kochwa and de Vries (1960) studied 58 human patients with urate calculus disease and observed that the morning urine was unusually acidic (pH 5). Henneman, Wallack and Dempsey (1958) suggested that the responsibility for urate calculus formation lay in a metabolic defect in which insufficient ammonia production in the renal tubules created a low urinary pH. In the cases observed in the Dalmatian, however, none has shown an abnormally low pH and in urines taken at operation the pH has been very high, and indeed abnormally high ammonia levels have been observed. This increase is possibly due to inflammation and infection. Infections, with the presence of urea-splitting organisms, have been regarded as important factors in stone formation since the organisms by producing ammonia increase the ~lkalinity of the urine, thus predisposing to the precipitation of phosphates. Whilst uric acid becomes more soluble with increasing pH, ammonium urate under such conditions would become less soluble due to the increased ammonium ion concentration: hence there would be an increased tendency for the precipitation of ammonium urate which is in fact the least soluble salt of uric acid. This would possibly explain the observation that urates and phosphates are frequently mixed in calculi. The urinary pH rapidly returns to normal after removal of the calculi. Dietary factors are considered to be important in the production of calculi. Vitamin A deficiency has been cited as a factor in stone formation mainly because of the experimental production of calculi
134
URATE STONES AND PURINE METABOLISM
in rats by Higgins (1951) with a vitamin A deficient diets Dalmatians on a diet deficient in Vitamin A also formed uric acid stones. It has been considered that a necessary measure in prevention of urate calculi is the feeding of a low purine diet. Brewer (1954), in treating a case of uric acid stones in a Dalmatian, suggested such foods low in purines as cheese, eggs, milk, cereals, fruit and vegetables and advocated that fats should be avoided, but Young, Conway and Crandall (1938), in metabolic experiments using synthetic diets, found that though the uricolytic index varied with the diet and the individual Dalmatian, there was no conclusive evidence for a particular dietary precursor in the synthesis of uric acid. Guanine, xanthine, uric acid and nucleic acids administered in capsule form increased the output of allantoin and urea but not of uric acid, and the excretion of uric acid was so constant that they suggested it was an endogenous metabolite. On the basis of these experiments there would thus appear to be little value in controlling the purine intake. The control of urinary pH by feeding alkaline salts such as potassium citrate could be of value, but the indications are that the problem of urate stones in the dog is different from that in man. The effect of the ammonium ion concentration, in the first instance, would appear to be more important than the pH in influencing urate calculus formation in the dog, and though an increase of pH would certainly increase the solubility of uric acid it would have no effect on the solubility of ammonium urate which is a poorly dissociated salt. Experiments to evaluate the efficiency of alkaline salts in the control of urinary ammonium ion concentration will be reported later. Alkalinisation of the urine may result in other complications, however, since it involves the danger of inducing the precipitation of phosphates. These observations emphasise the need for further study of the factors predisposing to the precipitation of urates and this forms the subject of further papers. CONCLUSIONS
Analysis of urate calculi from 17 cases in dogs using a microchemical technique, paper chromatography, ultra-violet and infrared spectrophotometry has shown that the main constituent is ammonium urate, and that some phosphate is usually present as well. Urate stones were found mainly in the Dalmatian (12 cases) but also in other breeds (5 cases). The determination of uric acid and allantoin in urine samples d h h . I . . d (AllantoinN 2 ) . d' lcate t att euncoytlcIll ex U· . N 2 XIOO nc A Cl'dN 2 +AII antOln of the Dalmatian was 36'3 ± 10'9 and of other breeds 92'4 ± 6'02. Although individuals in other breeds may excrete urate within the lower limits found for the Dalmatian they only do so by simultaneously excreting high concentrations of allantoiD., i.e. they have a high uricolytic index. III
P. PORTER
135
The concentration of uric acid in the urine of calculus-forming Dalmatians was no greater than in normal dogs of this breed and the level of uric acid in the urine does not appear to be the important factor in urate calculus formation. In contrast to the position with uric acid calculi in man, the urine in Dalmatians with urate calculi was not unusually acidic. The ammonium ion concentration, however, appears to be an important factor in urate calculus formation in the dog. ACKNOWLEDGMENTS
I am indebted to all the veterinary surgeons and dog breeders who supplied calculi, urines and details of the animals concerned, to Professor E. G. White for his constant interest and advice, and the Wellcome Trust for a grant which covered the cost of much of the work. REFERENCES
Atsmon, A., Frank, M., Lazebnik, j., Kochwa, S., and de Vries, A. (1960).]. Uro!.,84, 167. Benedict, S. R. (1916).]. Lab. clin. Med., 2 I. Brewer, N. R. (1954), N. Amer. vet., 35, 372. Brodey, R. S. (1955).]. Amer. vet. 'II1ed. Ass., 126, I. Dent, C. E., and Philpot, G. R. (1954). Lancet, 266, 182. Dikstein, S., Bergmann, F., and Chaimovitz, M. (1956). ]. biol. Chem., 221,239· Friedman, M., and Byers, S. O. (1948). Ibid., 175, 727. Gudzent, F. (1908). Z. physiol. Chem., 56, 150. Henneman, P. H., Wallack, S., and Dempsey, R. F. (1958). ]. clin. Invest., 37, 901. Higgins, C. C. (1951). ]. Amer. vet. med. Ass., lI8, 81. Hunter, A., Givens, M. H., and Guion, A. N. (1914), ]. biol. Chem., 18, 387. Keeler, C. E. (1940).]. Amer. vet. med. Ass., 96,507. Klemperer, F. W., Trimble, H. C., and Hastings, A. B. (1938). ]. biol. Chem., 125, 445. Larson, H. W. (1932). Ibid., 94, 727. Praetorius, E. A., and Poulson, H. E. (1953)' Scand. ]. clin. Lab. Invest., 5, 273. Prien, E. L., and Frondel, C. (1949).]. Urol., 57,949. Trimble, H. C., and Keeler, C. E. (1938).]. Hered., 30,281. Vischer, E., and Chargaff, E. (1948).]. bio!. Chem., 176, 703. Wells, H. G. (1918). Ibid., 35,221. White, E. G. (1944),], compo Path., 54, 16. White, E. G., Treacher, R. j., and Porter P. (1961). Ibid., 71.,201. Wolfson, W. Q., Huddleston, B., and Levine, R. (1947). ]. clin. Invest. 26, 995. Young, E. G., Conway, C. F., and Crandall, W. A. (1938). Biochem. ]., 32, 1138. [Received for publication, October 22nd, 1962]