Henry Ford Hospital Medical Journal

Volume 19 | Number 2 Article 9

6-1971 Observations on Transport in Man, the Dalmatian and the non-Dalmatian Howard Duncan

April S. Curtis

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Recommended Citation Duncan, Howard and Curtis, April S. (1971) "Observations on Uric Acid Transport in Man, the Dalmatian and the non-Dalmatian Dog," Henry Ford Hospital Medical Journal : Vol. 19 : No. 2 , 105-114. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol19/iss2/9

This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. Henry Ford Hosp. Med. Journal Vol. 19, No. 2, 1971

Observations on Uric Acid Transport in Man, the Dalmatian and the non-Dalmatian Dog*

Howard Duncan, M.D.** and April S. Curtiss, B.S.**

The urinary output of uric acid from the purebred Dalmatian is similar in amount to that in man. Also, this breed of dog has a higher plasma uric acid level than other and, like man, this is accompanied more frequently with renal and bladder lithiasis. AUopio-inol is effective therapy in both man and dog. Study of the fate of uric acid in the Dalmatian shows that the liver does not oxidize the available uric acid completely although it is capable of doing so when liver homogenates are studied. Consequently, the hepatic cellular membrane appears impermeable, or partially so, to uric acid. The pos­ sibility of a general membrane transport problem similar to that encountered at the liver cell has not been confirmed with studies of red cells in the Dalmatian. This dog shows some similarities to certain rare clinical human diseases with deafness, cardiac arrhythmias and renal tubule leak of uric acid, all of which offer ample opportunity for close and detailed examination as clinical models in biochemical, physiological, pathological and genetic studies.

Uric acid, the end product of purine clinical hazards. However, once a pa­ metabolism in man, and also the higher tient has been identified as a former of apes, is a highly insoluble and biologi­ uric acid stones, particularly in gouty cally benign material. It introduces a or with primary hyperuricemia, problem of excretion because its solu­ therapy aimed at correcting these prob­ bility in water at 40°C is only 50 mg lems has to be continued indefinitely. per liter. In urine the solubility is Man is not as fortunate as the lower slightly greater and the concentration animals, which are capable of handling is usuafly near saturation level. The uric acid in a more efficient manner. clinical problems of uric acid stone Further down the evolutionary scale, formation and obstructive uric acid the fish and amphibia excrete most of nephropathy develop as a consequence the nitrogen of protein metabolism as of this limited solubility. Uricosuric urea and ammonia and the mammals drugs (probenecid, salicylates, sulfin­ have also maintained this mechanism pyrazone, etc.) and more recently al­ as a major means of nitrogen excretion lopurinol (an enzyme inhibitor of uric (Fig 1). However, the reptfles and the acid oxidation) for the first time have birds developed a very clever water- successfufly controlled many of these conserving system whereby the last phases of breakdown from protein ni­ '•'Support for these studies was made pos­ trogen to urea were eliminated, and sible by grants from the Michigan Chap­ uric acid is the end-product. This ter of the Arthritis Foundation and Henry Ford Hospital (Grant #437). allowed twice the number of nitrogen • "•'Division of Rheumatology atoms to be excreted per molecule of

105 Duncan and Curtiss

available water. In the bird the plasma converts the insoluble uric acid to the uric acid level is simflar to man (3-7 more soluble allantoin (Fig 1). Man, mg/100 ml) and the uric acid is con­ however, has not shared this bequest centrated three-thousand times in the from aquatic ancestors. Partly because tubular urine of the bird and secreted of these basic metabolic differences into the cloaca.^ From here, the water between man and dog, research into is reabsorbed by the cloacal membrane, the control of the uric acid problem and the uric acid in solution, already has been slow. These biological differ­ supersatured crystalizes and, with this, ences result in man having a serum becomes osmotically and finally bio­ uric acid level of around 3-7 mg/100 logically inactive. The insolubility of ml and he excretes some 500-700 mg this white uric acid paste can be seen of uric acid per day in the urine while on the superior aspects of our public the dog with a plasma uric acid level buildings and statues, which have been of less than 0.3 mg/100 ml has a daily visited by pigeons and starlings. 24-hour urine output of uric acid of In the dog and similar lower mam­ 10-60 mg. Consequently, the solubflity mals uric acid is effectively oxidized problems for the dog are negligible and by the hepatic enzyme uricase, which uric acid lithiasis is rare.

PATHWAY OF PURINE DEGRADATION \ GLYCINE NUCLEOPROTEIN

PURINES

ADENOSINE GUANOSINE MAN, APES FISH HYPOXANTHINE * BIRDS MAMMALS i REPTILES XANTHINE I ./ URIC ACID _ i ALLANTOIN \ UREA, AMMONIA Figure 1

106 Uric Acid Transport in Man, the Dalmatian and the non-Dalmatian Dog

The purebred Dalmatian (Fig 2) is nize its intermediate position, in the distinguished biochemically by its ex­ handling of uric acid, with man on the tremely high uric acid output (400-600 one hand incapable of oxidizing uric mg/24 hours in a 10 kgm dog). The acid and on the other the non-Dalma­ plasma uric acid level is two to four tian breeds oxidizing uric acid most times that of the mongrel dog and as a efficiently. For this and other reasons consequence the Dalmatian* pays the to be enumerated, this purebred dog penalty for his genetic abnormality serves us with a remarkable oppor­ with a very high frequency of renal and tunity to study the physiological, bio­ bladder stones. During the past four chemical and pharmacological actions years we have used allopurinol in the and reactions of uric acid. Even Dalmatian to control the uric acid though this biochemical attribute was production and reduce the uric acid recognized specifically in 1916,- the output in the same manner as we have amount of such investigation has been in humans with the same problems. singularly little. Only in recent times In this particular breed we recog- has there been sufficient interest in this particular breed to bring about the '•'The reason that the dog has been called by this name is uncertain. There is no establishment of the Dalmatian Re­ reason to believe that it derived from search Foundation in 1969.** Dalmatia (Yugoslavia) anymore than it Aside from the basic uric acid dis- was first bred in England or Bengal al­ though it has often been called an English "•'Through the efforts of a Dalmatian fan­ Coach Hound and the Bengal Harrier. cier: J.C. Lowery of York, Pennsylvania.

ES

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Duncan and Curtiss

turbances, there are other areas of deaf Dalmatians, it was noteworthy physiological, pathological, clinical and that this "syndrome" in the Dalmatians genetic significance known to be more resembled an unusual clinical picture common in this breed than others. also seen in man. In this syndrome, The main biological aberrations in offspring of cousin marriages developed the Dalmatian include: combinations of congenital deafness, (1) Unique urate metabolism in: pigment patches, bizarre electrocardio­ a. liver grams, renal disease, and sudden b. renal clearance death. To our knowledge, uric acid of (2) Congenital deafness these children is not abnormal. (3) Cardiac arrhythmias and arrest The deafness in these dogs is re­ (4) Heterochromia of the iris, china ported to be due to Vlllth nerve dam­ eye and walleye age, but degeneration of the organ of (5) Renal calculi, hematuria, pyelo­ Corti and coflapse of the cochlear nephritis duct and saccule have been described.'' (6) Pigment "patches" These defects seem unrelated to the (7) Recurrent dermatitis uric acid abnormality. While many of these characteristics Although the full uric acid story is are also seen in other pure-bred dogs not yet known, it is clear that in the and reflect the result of close inbreed­ Dalmatian at least two distinct ab­ ing—the uric acid disturbance is normalities exist. In the mongrel dog unique to the purebred Dalmatian. The and in the human, uric acid is filtered high level of uric acid in the urine and through the glomerulus and approxi­ plasma falls dramatically in the off­ mately 98%-100% of this is reab­ spring of a cross between a pure Dal­ sorbed by the proximal tubules and matian bred to any other strain. Even that which ultimately reaches the urine by careful "back breeding" of such is thought to be almost entirely secreted offspring into the original pure strain, by the distal tubule (Fig 3). In the Dal­ the uric acid abnormality reacts as a matian, there is no resorption of uric true recessive; even though the charac­ acid at the proximal tubule and a small teristic polka-dot pattern of the coat amount is secreted by the distal tubule. can be reconstituted, the uric acid ab­ Hence in the Dalmatian, the uric acid normality fails to reappear.3 The es­ clearance may be equal to or even sence of this study reveals that the greater than the creatinine and insulin spotting phenomenon of the Dalmatian clearances in the same animal.^ * and the uric acid abnormality are This renal abnormality in the Dal­ coded on different genes. matian aflows the uric acid to be In 1967, James,* at the Henry Ford rapidly excreted and would tend to Hospital reviewed the cardiograms of produce the low plasma uric acid level 32 normal and 9 deaf purebred Dalma­ (0.5-1.0 mg) seen in this dog compared tians and found a higher incidence of with that of man as we noted earlier. cardiac arrhythmias in the deaf dogs The high urine uric acid output from compared with the normals in the same a 10 kgm dog equals that of a 70 kgm breed. Whereas it is our experience man. A counterpart appears in human that the sudden death may occur in patients with Wflson's disease or hepa-

108 Uric Acid Transport in Man, the Dalmatian and the non-Dalmatian Dog

URATE TRANSPORT IN THE KIDNEY

Non-Dalmatian Dalmatian Man

Glomerulus 100: No 98% reabsorption reabsorption reabsorptioirption ^ secretion Proximal 3% 5% secretion tubule

±3% •'secretion 5% Distal ^ +3% 'secretion Tubule secretion

3% 110% 10% estimated filtrate estimated filtrate estimated filtrate

Figure 3 tolenticular degeneration with a modi­ to the renal differences mentioned, the fied deToni Fanconi syndrome wherein Dalmatian fails to oxidize uric acid as the proximal tubules fafl to resorb uric efficiently as the mongrel. In all dogs acid and the uric acid clearance ap­ uric acid is oxidized by the enzyme proaches that of creatinine. In these uricase which is abundantly present in young people it is usually noted that the liver including that of the Dalma­ the serum uric acid is in the range of tian. There is no significant amount of 0.5 to 1.5 mg/100 ml. If the Dalmatian uricase or other uric acid oxidizing kidney is capable of excreting 10-100 enzyme in any other tissue.^ Several times more uric acid per day in the studies have shown that the hepatic urine than the mongrel dog, why then oxidation of uric acid in the Dalmatian is its plasma uric acid level 3-4 times differs from that of the non-Dalmatian. higlier rather than lower than that of One series of observations^" showed the mongrel dog? It is important to that when the ureters of the mongrel understand at the same moment that dog were clamped, the serum uric acid there is no evidence that there is in­ level remained low despite the increas­ creased nitrogen turnover in the Dal­ ing levels of creatinine and other nflro- matian compared with the non- genous end-products. This was felt to Dalmatian dog. This being so, some be due to efficient oxidation of the other aberration in the metabolism of excess urate load by hepatic uricase. uric acid must exist in this animal. In the Dalmatian the serum uric acid It has been shown that in addition level rose rapidly together with the

109 Duncan and Curtiss serum creatinine and urea when the ciendy as the mongrel dog. same studies were performed. Another Observations: Eight Dalmatians and study'^ showed that the liver could be six mongrel dogs were anesthetized implicated in the Dalmatian's faflure and small cannulae were placed in each to oxidize an excess intravenous load of the following sites: (1) abdominal of uric acid. The evidence was that al­ aorta; (2) renal vein; (3) portal vein; though the intact kidney excreted 52% (4) hepatic vein; (5) jugular vein. After of the intravenous load in 60 minutes, baseline blood samples were taken the plasma uric acid level was stfll six from these sites, a rapid injection of times the normal figure whereas a uric acid (40/mg/kg) was given into simflar load given to non-Dalmatians the jugular vein. At intervals of five was completely oxidized within 60 minutes after this bolus injection and minutes—even though only 12% of for a period of 40 minutes, samples the load was recovered in the urine and were drawn simultaneously from each the plasma level was normal. These site cannulated and uric acid levels studies appeared to confirm the prem­ were determined on each sample ise of Klemperer et aP who in 1938 (Table 1). The arteriovenous urate dif­ stated that the Dalmatian liver con­ ferences for liver, kidney and gastro­ tained uricase which was quantitatively intestinal tract were calculated. capable of oxidizing as much uric acid From these studies two important as the mongrel dog, but the liver cell points became evident: (1) The hepatic wafl in some way prevented this action. "clearance" of uric acid in the mongrel His evidence depended upon the astute dog was greater than the Dalmatian observations that when liver tissue for, in the latter, the arteriovenous from Dalmatian and from non- difference was similar for all organs Dalmatian dogs was homogenized and excepting the kidney. (2) There was incubated with uric acid, the rates of no measurable loss of uric acid into oxidation of uric acid by these homo­ the gastrointestinal tract in eflher genates was the same for each group. group. (This fact is rather significant However, when intact liver slices were when it is remembered that in humans used in the same type of uric acid some 200-400 mg of uric acid per 24 oxidizing system, the slice of non- hours is secreted into the gastroin­ Dalmatian liver was greatly superior testinal tract and is oxidized.) From in oxidizing uric acid than the slice this, we deduced that the circulating of Dalmatian liver. The inference from uric acid in the Dalmatian is prevented these studies was that the intact hepatic from being oxidized presumably by ceflular membrane prevented the as­ some block in the membrane perme­ sociation between uric acid in the ability to uric acid in the Dalmatian blood and the uricase present in the hepatic structure. hepatic cell. If such a problem exists in one In recent studies we have shown that region of the purebred dog, does this although a constant load of uric acid account for the differences in handling is delivered to the Dalmatian liver, uric acid in the renal tubule and might both by hepatic artery and portal vein, it not be present also in some other it fails to oxidize the uric acid as effi- more accessible areas such as the red

110 Uric Acid Transport in Man, the Dalmatian and the non-Dalmatian Dog

TABLE I

Mean Values* of Serum Uric Acid (mg/lOOmI) Determined at Intervals Following l.V. Injection of 40 mg/kg of Uric Acid

NON-DALMATIAN DALMATIAN Time (minutes) Renal Vein Hepatic Vein Renal Vein Hepatic Vein

0 0.6 0.6 0.4 0.8 5 8.1 5.8 12.2 15.1 10 4.1 3.1 10.0 11.1 15 3.8 2.4 8.3 8.6 20 3.6 1.8 7.4 7.8 30 2.9 1.6 5.6 6.8 40 1.0 0.9 5.0 5.4

* Eight Dalmatians and six mongrel dogs were studied blood cell or the white blood cefl? In mediated transport is blocked by the humans the polymorphonuclear leuco­ presence of other purines, i.e., hypox­ cyte is capable of concentrating uric anthine, and accelerated in the pres­ acid—a situation which is occasionally ence of estrogens. We have conducted seen in gouty patients whose peripheral such studies on 40 normal adult and circulating polymorphonuclear cells'^ gouty patients in an endeavor to deter­ contain one or more large crystals of mine the presence of urate transport uric acid. abnormalities. No such discrepancy Uric acid is also present wflhin hu­ has been noted. man red blood cells and Danish work­ By using this system on two Dalma­ ers have studied the transport of uric tians and "a number" of mongrel dogs, acid across the red cefl membrane.'^•i- Harvey and Christensen^^ suggested They have shown that although physi­ that a red cell membrane transport cal diffusion of uric acid occurs, an block existed in the Dalmatian's eryth­ active transport system—"mediated rocytes and not in those of the mongrel transport"—accounted for the majority dog. Such a circumstance would help of transfer of uric acid from the sus­ explain the problem at the hepatic cell pending medium to the red blood cefl. membrane in the Dalmatian but, un­ This urate transport was altered by pH fortunately, we have not been able to changes, temperature changes, and confirm these studies even though per­ changes in concentrations to which the formed identically and repeatedly on Michaelis equation for enzyme reaction erythrocytes of 10 purebred Dalma­ rates could be applied. This enzyme tians and 14 mongrel dogs.

Ill Duncan and Curtiss

Method tion of intracellular radioactivity sep­ The detafls of the method have been arately. While normal physical dfliu- previously delineated.'Basically the sion occurs, evidence of the presence movement of uric acid across the red of a mediated transport system is ap­ cell membrane is monitored by using preciated by the more rapid fall in the radioactive uric acid (labelled '^C in radioactivity of the supernatant fluid. the "2"-position). In the steady state If hypoxanthine is added to the incu­ the levels of uric acid within the cell bating medium, mediated transport is and in the suspending medium do not stopped and the slow decline in the change but a steady influx and efflux radioactivity of the supernatant repre­ of uric acid molecules is constantly oc­ sents physical diffusion factor alone. curring. The influx is measured by the rate of decline of concentration of la­ Results belled uric acid in the suspending Table 11 shows the percentage of medium during 60 and 120 minutes of the radioactive uric acid present at zero incubation. If hypoxanthine has pre­ time, 60 minutes, and 120 minutes viously been added to the medium, the foflowing its introduction to the sys­ active transport of uric acid is in­ tem. Human, Dalmatian and non- hibited. Dalmatian red cells have been studied. Fresh samples of blood were centri- Included also in the study on the super­ fuged and the red cells separated, natant are figures available for these washed, and suspended in a physiologi­ changes when hypoxanthine is present cal buffer containing a uric acid con­ in the suspending medium. In the hu­ centration equal to the previous plasma man the concentration of the labelled level. These cefls (5 ml) were incubated uric acid fefl from 100% at zero time with 5 ml of buffer at 24°C for 45 to 81.9% at 60 minutes and 75.3% minutes to permit a steady state to be at 120 minutes. When hypoxanthine established and then at zero time 0.4 was added, the mediated transport sys­ microcuries of uric acid in 0.11 mg of tem was blocked and the fall of radio­ stable uric acid contained in a 0.2 ml activity at 60 minutes was 89.3% and volume was added. Aliquots were at 120 minutes was 84.3% which taken immediately after mixing at zero represented the slow physical diffusion time, and at 60 minutes and 120 min­ factor. In the studies involving the sets utes and at intervals between. The cells of dogs no evidence of a mediated were rapidly centrifuged at 0°C in a transport of uric acid was found for two-minute period and samples 0.2 ml the studies in the presence and absence of the supernatant were transfered to of hypoxanthine were the same and scintiflation-counting vials. The trans­ also the same for each breed. Whereas fer across the red blood cell membrane a difference is known to exist between and into the red cell was represented by human and canine red cell transport, the fall of radioactivity of the super­ Harvey and Christensen stated that a natant fluid and can be cross-checked 13% difference between the mediated subsequently by separation of the red and the physical diffusion transport blood cells then promoting their he­ systems was evident in mongrel dogs molysis and evaluating the concentra­ but was not present in Dalmatians. In

112 Uric Acid Transport in Man, the Dalmatian and the non-Dalmatian Dog

TABLE II

Uric Acid Transfer into Red Blood Cells Percentage Radioactivity Present in Supernatant at Intervals Following Zero Time. (± Standard Deviation)

Medium 0 Minutes 60 Minutes 120 Minutes

Human Buffer only. 100 81.9 ±3.4 75.3 ±5.0 RBC's With hypo­ xanthine. 100 89.3 ±4.0 84.3 ±4.4

Mongrel Buffer only. 100 92.0 ±2.2 85.6 ±2.0 RBC's With hypo­ xanthine. 100 92.9 ±2.0 86.4 ±2.4

Dalmatian Buffer. 100 92.7 ±2.3 85.4 ±1.9 RBC's With hypo­ xanthine. 100 93.4 ±1.6 87.4 ±2.1 our own studies, no such difference maturia of Dalmatians should be sus­ was demonstrable. ceptible to the beneficial effects of al­ This method of observing the con­ lopurinol since nucleoprotein degrada­ centrating of labelled purines, by sus­ tion in both man and dog are similar. pended red blood cells, forms the basis Using seven Dalmatians and three of the present tests used in confirming mongrels which received 100 and 200 the diagnosis of the Lesch-Nyhan syn­ mg/day of allopurinol, our studies drome. In this syndrome, a partial or showed the average uric acid output of complete deficiency of the enzyme the Dalmatian could be reduced from hypoxanthin-guanine phosphoribosyl 580 to 420 mg per 24-hour period—a transferase presents itself clinically in 27% decrease. We have not conducted chfldren with mental deficiency, neuro­ any long term studies on the frequency logical abnormality, self-mutilating of stones in the dog population but one tendencies, hyperuricemia, and often can deduce that if uric acid load should uric acid lithiasis. In this group of pa­ be decreased, its associated problems tients allopurinol has proven partially also should be reduced. effective in reducing the hazard of uric The uric acid problems in man are acid stone formation. beginning to be understood and while In the gouty patients and idiopathic some uric acid studies on the chim­ uric acid stone formers, allopurinol has panzee have been recently described^' proven eminently effective. Here, which show a very close simflarity to again, it would naturally follow that man, the Dalmatians' abnormaUties are the uric acid stone formation and he­ much more readily accessible and per-

113 Duncan and Curtiss mit the understanding of uric acid me­ faster and more economical rate with tabolism and membrane transport mutual benefit to both man and dog. problems in man as well as the other Acknowledgement aberrations found in this dog and Considerable technical help is ac­ other purebreds. A closer attention to knowledged from the Surgical Re­ this breed with respect to the study of search personnel and particularly Mary biochemistry, physiology, pathology, Jo Lassflla, Jerry Mackenzie, Vladimir and genetics could be productive at a Sadler, and W. M. Konde, D.V.M.

REFERENCES 1. Smith, H. W.: From fish to philosopher, Boston: Little, Brown and Co., 1953. 2. Benedict, S. R.: Uric acid in its relations to metabohsm, Harvey Lectures 11:346-65, 1915-16. 3. Trimble, H. C. and Keeler. C. E.: Inheritance of hish uric acid excretion in dogs, / Hered 29:145-8, 1938. 4. James, T. N.: Congenital deafness and cardiac arrhythmias, Amer J Cardiol 19:627-43, May 1967. 5. Hudson, W. R., and Ruben. R. J.: Hereditary deafness in the Dalmation dog, Arch Otolaryngol 75:213-9, Mar 1962. 6. Kessler, R. H.; Hierholzer, K., and Gurd, R. S.: Localization of urate transport in the nephron of mongrel and Dalmatian dog kidney, Amer J Physiol 197:601-3, Sept 1959. 7. Lassen, U. V.: Kinetics of uric acid transport in human erythrocytes, Biochim Biophys Acta 53:557-69, 1961. 8. Yu, T. F., et al: Tubular secretion of urate in the dog, Amer J Physiol 199:1199-204, 1960. 9. Klemperer, F. W.; Trimble, H. C, and Hastings, A. B.: The uricase of dogs, including the Dalmatian, J Biol Chem 125:445-9, 1938. 10. Friedman, M., and Byers, S. O.: Observations concerning the causes of the excess excretion of uric acid in the Dalmatian dog, J Biol Chem 175:727-35, 1948. 11. Duncan, H.; Wakim, K. G., and Ward, L. E.: The effects of intravenous administration of uric acid on its concentration in plasma and urine of Dalmatian and non- Dalmatian dogs, / Lab Clin Med 58:876-83, Dec 1961. 12. Lassen, U. V., and Overgaard-Hansen, K.: Purine derivatives as inhibitors of uric acid transport into human erythrocytes, Biochim Biophys Acta 57:118-22, 1962. 13. Harvey, A. M., and Christensen, H. N.: Uric acid transport system: Apparent absence in erythrocytes of the Dalmatian Coach Hound, Science 145:826-7, Aug 1964. 14. Fanelli, G. M., Jr.; Bohn, D. L., and Reilly, S. S.: Renal urate transport in the chimpanzee, Amer J Physiol 220:613-20, Mar 1971.

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