Veterinary Research Geo Abstracts Ltd,

FOOD

INTAKE

AND

Coizimmications, Norwich - printed

STRUVITE

11 (1987) 519-526 in England

CRYSTALLUKlA

VJ. P. PALMORE ix K. II. BAkTOS Department of Physiological Sciences, Gainesville, Florida 32610, USA

J-144

IN

519

FERRETS

JhlvlHC,

University

of

Florida,

This research was supported in part, by Grants F\lo. BKSG 5uscl7 KliU7UZl-14 (DSR) and IjKSG KKO57M3-03 (CVM). Florida Ayriculture Experiment Station Journal Series No. jXI5 (Accepted:

5 March

lVB7)

ABSTKACT Palmore, ferrets.

W.P. & Bartos, K.D. 1987. Food Veterinary Research Communications,

intake ll(6j.

and struvite 519-526

crystalluria

in

Four adult, castrated, male ferrets were studied in two similar trials for effects of food intake on variables hypothesized to promote struvite (ammonium, magnesium, phosphate hexahydrate) crystal formation in urine. Struvite crystalluria occurreu in three of the four ferrets. Urine ph (Upl-i) averaged 6.6 for these ferrets. Uph in the ferret without crystalluria was 6.0. By simple linear regression analysis, no relationship was found between the amount of food ingested ano the urinary concentration and excretion of magnesium and phosphorous. However, osmolality and urine excretion of both protein anu ammonium were correlated to food intake (P( .U5j. Ways in which these effects coulo promote struvite crysLa1 formation are discusseo.

Ferrets are susceptible to a form of struvite urolithiasis (SU) not accounted for by the urine alkalinizing, ammonium il\lH4)-generating effects of urease-positive rnicrobiai infections that are commonly responsibie (Griffith & Musher, 197b; Nguyen et al., 1979; i3ircn et al., 1981j. While the pathogenesis of tnis form of SU is poorly understood, one or a combmation of three mam hypotheses for urolith forrnation may be operative (Wiiliams, 19743. The hypotheses are: the crystalhzation-precipitation hypothesis; the matrix-nucleation hypothesis; one the inhibitor-absence hypothesis. These hypotheses, a!though funoamentally different in many respects, have the common requirement that conurtions in the urinary tract be supportive of crystal formation. Consequently, environmental factors that change these conditions are likely to be important in the patnogenesis and prevention of SU. Because epidemiological evidence suggests tne nature and the alhount of food ingested is one such factor
ANO

METHODS

Four aault, castrated, male ferrets, Iviustela putorius furo, were stuuied in two similar trials conoucted 111 days apart. Before the trials oegan the ferrets were adapteu to the husbandry ano feediny regime for at least one 0165-7380/87/$03.50

c

1987 Gee Abstracts

Ltd

520

Inonth. They were kept in rabbit metabolism cages in an air-conditioned room with a 12 hr 1ight:aark cycle throughout the study. The cages were cleaned thoroughly at 8 am each day. Food and water intakes also were measured. After subtracting the 12% proportion of the ration that was water from the total food intake, the weight of the food ingested was recorded. The amount .of water ingested in the ration was added to the water intake values. Halston-Purina Lat, Canine Diet (Fialston-Purina Co., St. Louis, MO) and water were provided ad Iibitum except for the last day of each trial during which water was removed for 20 hrs. The feed label indicated the diet contained not less than 25% crude protein or more than 9% crude fat, 4% crude fiber, and 12% moisture. The elecLrolyte content of the ration was measured. For this, samples of the oiet were digested in hot nitric acid as recommended by tiorwitz (1980). Sodium (Na), and po&;yiurn (Kj content wa; deterrnined oy flame photometry; magnesium and phosphorous (PI content was measured by atomic absorption spectfophotometry in the University of Florida, Institute of Footi and Agricultural Science Research Support Laboratory. The ration contained on a dry weight basis 0.48% Na, O.aiL/o K, 0.16% Ivig, and 1.0% P. Urine was collected at 6 am and 4 pm each day. The divided urine samplings within each 24 hr collection were necessary to meet the urine preservation requirements for different analyses. Specifically, acidification of one sample was necessary to prevent spurious urinary ammonium (UNH4) measurements due to spontaneous losses of amrnonia (NH3), gas from the sample. Another sample, maintainea at the pH of freshly volded urine, was required Lo detect struvite crystals since they dissolve in the acid solutions necessary for NH4 analysis. To accommodate these requirements, II.5 ml of concentrated HCI were aaded to the collecting vessel for the 8 hr sample, ana 4 drops of 10% formalin were added to the collecting vessel for the 16 hr sample. Formalin preservation of urine aoes not change urinary pH (UpIdirectly and prevents bacterial growth (Ames Laboratories, 1982). Moreover, Uph, urinary protein (Uprot), and most other urinary solutes do not change with time in formalin-preserved urine. A few drops of mineral oil added to both collection vessels prevented evaporative losses from the surface of samples. Urine was collected from the bottom of the metabolism pans in 50 ml syringe barrels converted to function as separatory funnels with a 3-way stop-cock. By draining the urine through the stop-cock, mineral oil contamination of samples was avoided. Contamination with food and faeces did not occur because the ferrets habitually defecated in one corner of the 51 x 56 cm cage walk floor, anti neither food nor faeces passeo through the small-mesh grating. Amrnonium excretion was determined from the 8 hr urine samples; the 16 hr sample was utilizeo for the microscopic exanlination of urinary sedirnent and all other urine analyses. These included analyses of pH (UpH), 149 (Urngj, P (Up>, protein (Uprot), and urea (Uurea). Ammonium concentration in urine (UNH4) was measured by the berthelot reaction as modified uy Chaney and Marbach (1962). Protein was measured by a dye-binding assay @radford, 1976). Urinary urea was measured by the diacetyimonoxirne method (Cracker, 1967). For measurements of Urng and Up an aliquot of urine was obtained from the formaiin-preserved sample after thorough mixing. The aliquot was adjusted to ph 1 by titration with concentrated hCI to assure struvite crystal dissolution and ionization of the constituent IbIg and phosphate. Samples were preparea for atomic absorption analysis as describea by Weissman and Pileggi (1974). Pnosphorous was measured by the Fisk-Subbarow

521

method using Sigma Chemical Co, St Louis, MO, Kit no. 67U. Urine ph was measured with a ph electrometer immediately after collection of the urine. Urine osmolaiity (Uosrn) was determined with the aid of a Wescore ivlodel 51008 Vapor Pressure Osmometer. The existence of relationships between variables was determined with simple linear regression; the degree of correlation is expressed by correlation coefficients (r) (Godfrey, 1986). Values from measurements made on successive days were averaged ano are reported as such. By averaging the data, the problem of inaccurate balance measurements due to the failure of animals in metabolisnt cages to empty the urinary bladder on an exact 24 hr schedule is reduced. For the first trial values were obtained over two days, and for the second trial over three days. Samples used for measuring the effects of water deprivation on Uosm were obtained the following day. Urine sediment was obtained from urine samples maintained at 37OC by centrifugation at 1500 g for 10 min. After the supernatant was aecanted, the sediment was mixed in the few drops of urine that adhered to the sides of the tube. The resultant mixture was examined microscopically for the presence of ‘coffin lid-shaped’ struvite crystals. RESULTS Struvite crystais were found in urine from three of the four ferrets. The UpH of these ferrets averaged 6.65 2 .Xi (mean ? sd), while the Uph of the ferret without crystalluria was 6.00. Despite the presence of crystals, there was no evidence of SU formation either during the experiment, or at necropsy when the experiment was ended. No simple relationship existeu between the presence of struvite crystals and Umg, UluH4 or Up either individually or collectively as determineo by relating the presence of crystals to the multiplication product of the tnree concentrations. Although UNH4 excretion and presence of struvite crystals were not oetermined in the same samples, there was no evidence that they varied independently of each other. However, such a dichotomy is possible, and the data are qualified to this extent. When the ferrets were deprived of water for 20 hrs, Uosm averaged 2365 2 126 m&m/kg of water (mean 2 sem). With water available ad libitum There was no correlation between Uosm was 1916 _+ 70 mOsm/kg of water. the renal concentrating capacity, as measured by Uosm, and the urine concentrations of NH4, Mg, P, and protein. Uosm was, however, correlated to Uurea (r=.94; P < .Ol). Based on scattering of nibbled food pellets from the food containers during the. work day and the rernaining food in the cage each morning, the ferrets apparently ate throughout the day. The amount of fooo the ferrets ingested was correlated to Uosm (r=O.tll; LP < -03) (Figure i-A) and to the amounts of Nh,,, protein, and urea excreted in the urine (rAI.89; P < .Ol; r=U.88, P < Al; r=.89; P < Al, respectively) (Figure l-B&). Correlation also existed between the amounts of protein ano hlg excreteo (r=0.81, P< .O>) (Figure 1-U). Correlation between the amounts of protein ana P excreted, though not statistically significant (r=U.75, P < .06), suggests a trend (Figure 1-E). DlSCUSSlON The goal of this study was to determine whether urine physico-chemistry varies with the amount of food ingested in ways that might promote struvite crystaliuria in ferrets. Potentially important relationships were detecteu but

522

Figure

1.

A. Correlation of urine osmolality to food intake. B. Correlation of urinary protein excretion to food intake. C. Correlation of urinary arnmonium excretion to food intake. 0. Correlation of urinary maynesium excretion to urinary protein excretion. E. Correlation of urinary phosphorous excretion to urinary protein excretion. Oata derived from two similar trials conducted 1U days apart. The lower case letters, a & b, on the face of the graphs identify each trial. lNumbers preceeding the letters identify individual ferrets. For trial ‘a’, each point is the average of measurements made on two successive days; trial ‘b’ each point is an average of three measurements.

523

complexities of given the oiomineralization ilowenstam, 1YSlj other relationships are probably also important. These could include those associated with post-prandial, circadian rhythm ano other types ot fluctuations in urine composition. Notwithstanding such reiationshrps the positive findings of the present study might promote crystalluria as follows. Theoretically, as water is removed tram the glomerular filtrate during the urinary concentrating process, Mig, PU4, and NH4 concentrations could become increasingly higner until the struvite solubility product is reached and crystallization occurs. However, crystailization uy this mechanism apparently did not occur, since no reiationship between Uosrn and either the concentrations or total amounts of these solutes in urine was found. Alternatively, Uosm may reflect a species-relateo risk rather than a dietrelated risk for crystal formation. Compared to hurnans in which maximum Uosm is approximately 1illG \Schmiot-Nielsen, 1977j, the ferrets proouced urine averagrng 1916 mOsm/kg of water when water was avaiiable ad libitum and 2365 m&m/kg of water during hydropenia. Interestingly, other species, such as cats and sheep, that are susceptible to the non-infectious form of SU also are capabie of producing urine of relativeiy high osmolality ?SchmidtNielsen, 1977; Palmore eL al., 1978). Hence, the high renal concentrating capacity, could be involved in struvite crystal fortnation even though the mechanism is not presently apparenl. Urinary protein excretion Increased as fooa intake increasea while big and PO4 excretion did not. Yet, the two minerals were correlated to protein excretion. For a soiute such as protein to be simultaneously correiated to food intake and to other solutes which are not theniselves correlated to food intake is a provocative observation that has no simple explanation. Perhaps a more complex relationshrp exists between food intake and the excretion of Mg and PU4 than can be detected by linear regression. At any rate, the increase in protein excretion with food intake and the correlation of Lne excretion, excretion of minerals contained in struvite with protein is consistent with the hypothesis that Uprot serves as a nidus upon which struvite crystals precipitate C.Wiilliams, 19743. The lack of a relationship between food intake ano urinary excretion of Mg and PCJ4 is not in itself surprising. Although urinary excretion of these electroiytes, is related to the amount absorbed into the oloodstrealrl from the is frequentiy a dichotomy between ingestion and the amount gut, there absorbed into the biooustream (Alfrey, lY76j. Excretion of NH4, one of the solutes containea in struvite, varied with the amount of fcod ingestea. Interpretation of this result uepenus partly on the acid-base status of the animal because one renal adaptation to acid loads 1574). ‘&her) this occurs UpH decreases is increase0 NH4 prouuction (Pitts, ano struvite crysLa1 formation is likely to be inhioited despite an increase in UiuH4,
524

the infectious form of SU, viz, by simultaneous increases in UNH4 alld UpH (tiriPfith tx Musher, 1976). Only the site of the Nhq production and the H+ buffering differs. The over-all pathophysioloyy could be the same. Inherent in the present discussion is that the conditions in urine necessary for crystals of the diet rnay themselves be changed. crystallization is a case in point. Struvite the UpH was less than pH 6.5 regardless of

diet-dependent variables change to form. however, these effects The effect of UpH on struvite crystalluria was not found when other conditions.

In other trials involving the same ferrets out a different ration, the same relations between the amount of food ingested, Uosm, UNH4 concentrations, and Uprot existed, but struvite crystals were not found (Bartos, 1981). This result suggests either that the amount of food ingested is not related to struvite crystal formation, or that neightened activity of one variable, eg, H+, overrides the sirnultaneous, heightenea activity of others. The fact that UpH averaged only 6.0 ? .05 when the non-crystallogenic diet was fed supports the latter view. tiespite this ambiguity in the results, tne relations between Uosm, Uprot and UNH4 and the amount of food ingested are of sufficient potential importance in explaining why sorne individuals develop SU, while others do not, to merit attention. however, the difficulty, and probably the futility, of tryiny to describe the formation of struvite crystals in urine in terms of a single, universally applicable variable also is apparent. The experirnental design did not allow measurement of the effect of each dietary constituent on the observed changes in urine composition. However, protein p.robabIy had a key role. The correlations between food intake and urea excretion Uosm and Uurea are consistent with this and oetween conclusion since urea excretion reflects protein catabolism. Moreover, other studies show that increasing protein intake promotes both renal concentrating capacity and protein excretion (Hendrikx & Epstein, 1950; Neuhaus ZX Flory, 1975; Palrnore, et al., i978). The increase in protein excretion is likeiy to result from increases in the amounts of protein filtered by the kidneys since increased renal glomerular filtration rate is another physiologic response to protein ingestion (Brenner et al., 1982). The mechanisms by which oietary protein increases renal concentrating capacity is the subject of several reviews (Hendrikx & Epstein, 1958; Schmidt-Nielsen, lY77). CONCLUSION Since crystals formed when one ration but not another was fed, the present study indicates that in ferrets urinary struvite crystal formation is diet-related under some circumstances. The amount ot food eaten may be among tne contributing factors. Increases in renal concentrating capacity, extra-renal ammonia production, ano protein excretion could oe involved. If so, protein is probably tne responsible dietary constituent. REFEKENCES Alfrey, A.C. 1976. Disorders of Mg metabolisrn. Kenal and electrolyte disorders. (Little, Brown Ames Quality Assurance Product Evaluation fieport No 8-82. Ames Gvision, Miles Elkhart, IN 46515, USA

In: K.W. Schrier (ed), and Co., fiostonj, 223-227

Laboratories, Laboratories,

1982. Technical Inc. PO Box 70,

525

bartos, K.U. 1981. Effects of diet on urine (IviSc thesis, University of Florldaj Birch, i).F., Fairley, in urmary tract 5b-64

K.F. h Pavillard, oisease: ureaplasrna

in the ferret,

K.E. 1981. urealyticum.

Chancy, A.L. & hiarbach, urea and Nh4. Clinical

E.P. i962. Chemistry,

putorlus.

Unconventional bacteria Kianey International, i9,

Bradford, M&i. i476. A rapid and sensitive method for microgram quantities of protein utiliziny the principle binuing. Analytical Biochemistry, 72, &+8-Z% Brenner, B&i., Meyer, T.W. CC Hostetter, and tile progressive nature of kioney Medicine, 3l~7, 652-659

Mustela

T.h. 1982. oisease. New

tne quantitation of protein-aye

Dietary England

Modified reagents 8, 13ii-132

for

protein Journal

of

intake of

deterrnmation

of

Cracker, C.L. 1967. Rapid determination of urea nitrogen in serum without deproteinization. American Journal of Medical Technology, 33, 561-365 Fraser, C.L. dc Arieff, A.I. 1985. Hepatic Journal of Medicine, 313, 865-873 Godfrey, K. iYd6. England Journal

encephalopathy.

Sirnpie linear regression in medical of Medicine, 313, 1629-1636

New research.

Englano New

Griffith, D.P. ij, Gusher, D.M. 1976. Pathogenesis of infection stones. In: 0. Finlayson h V4.C. Thomas (ec;sj, Colloquium on renal Iithiasis. (University Press of Florida, Gainesville), S4-65 Hendrikx, A. %s Epstein, renal concentrating 145, 559-542

F.H. ablllty

i958. Eftect of teeding protein anu urea in the rat. American Journal of Physiology,

on

Horwitz, W. 1980. Minerals In feed by atomic absorption. In: Official methods of analysis of the association of Official Analyticalists. i3tn eaition, Association of tifficlal Analytical Chemists, i36 Lewis, L.D., iviorris, M.L. Jr & hand, syndrorne. In: Small animal clinical Topeka, Kansas), Y-l to Y-32 Lowenstarn, 1131

H.A.

iY81.

iviinerals

iv,.S. 1987. Feline urolocjical nutrition, (Mark lvlorris Associates,

formed

by organisms.

Science,

211,

1116-

Neunaus, 0.W. h Flory, W. iY75. The effect of dietary protein excreLion of alpha ZU, the sex-dependent protein of the adult Biochimica et Biopilysica Acta, 411, 74-W

on the male rat.

Nguyen, H.T., Moreland, A.F. C!XShields, &P. 1979. Urohthiasis (Ivlustela putorius). Laboratory Animal Science, 29, 243-245

in ferrets

Palmore, w.P., Gaskin, J.ivi. ci: Nicison, J.T. 1978. urine. Laboratory Animal Science, 26, >51-555 Pitts,

R.F. 1974. Excretion of arnlmonia. the kidney and body fluids, 3ra edition, Clllcago), 217-237

Effects

In: Physioloqy of IMedical

(Yearbook

of diet

on feime

Publistlers,

526

Keif,

J.F., i3ovee, K., Gaskell, CA., t3att, H&i. & Maguire, T.G. 1977. Feline ureteral obstruction: a case-control study. Journal - American Veterinary lLiedicai Association, 170, 132U-1324

Schmidt-Nielsen, B. 1977. Excretion in mammals: role of the renal pelvis the modification of urinary concentration and composition. Federation Proceedinqs, 36, 2493-1503 Stone, W.J., Balagura, S., Pitts, R.F. & Shurland, L.M. 1967. Diffusion equilibrium for ammonia in the kidney of the acidotic dog. Journal Clinical Investigation, 46, 1605GO& Weissman, iedsj, How,

N. 2~ Pileggi, V.J. 1974. Inorganic ions. Uinical chemistry: principles and technics, Hagerstown, ivlc)), 670-679

Willeberg, P. 1975. Veterinarmedicin, Williams, H.E. 33-38

1974.

Gets anu the feline 27, 15 Nephrohthiasis.

urological

New England

in

of

In: K.J. Henry et al 2nd edition. (Harper and syndrome. Journal

Nordisk of Medicine,

290,