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Journal of Animal Science - Animal Nutrition

The effect of dietary hydroxyproline and dietary oxalate on urinary oxalate excretion in cats1

 

This article in JAS

  1. Vol. 92 No. 2, p. 577-584
     
    Received: Dec 13, 2012
    Accepted: Nov 22, 2013
    Published: November 24, 2014


    2 Corresponding author(s): e.a.plantinga@uu.nl
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doi:10.2527/jas.2013-6178
  1. J. C. Dijcker*,
  2. E. A. Hagen-Plantinga 2,
  3. D. G. Thomas,
  4. Y. Queau,
  5. V. Biourge and
  6. W. H. Hendriks
  1. Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands
    Institute of Food, Nutrition and Human Health, Massey University, Palmerston North 4442, New Zealand
    Royal Canin Research Center, 30470 Aimargues, France
    Animal Nutrition Group, Wageningen University, 6700 AH Wageningen, The Netherlands

Abstract

In humans and rodents, dietary hydroxyproline (hyp) and oxalate intake affect urinary oxalate (Uox) excretion. Whether Uox excretion occurs in cats was tested by feeding diets containing low oxalate (13 mg/100 g DM) with high (Hhyp-Lox), moderate (Mhyp-Lox), and low hyp (Lhyp-Lox) concentrations (3.8, 2.0, and 0.2 g/100 g DM, respectively) and low hyp with high oxalate (93 mg/100 g DM; Lhyp-Hox) to 8 adult female cats in a 48-d study using a Latin square design. Cats were randomly allocated to one of the four 12-d treatment periods and fed according to individual energy needs. Feces and urine were collected quantitatively using modified litter boxes during the final 5 d of each period. Feces were analyzed for oxalate and Ca, and urine was analyzed for specific density, pH, oxalate, Ca, P, Mg, Na, K, ammonia, citrate, urate, sulfate, and creatinine. Increasing hyp intake (0.2, 2.0, and 3.8 g/100 g DM) resulted in increased Uox excretion (Lhyp-Lox vs. Mhyp-Lox vs. Hhyp-Lox; P < 0.05), and the linear dose-response equation was Uox (mg/d) = 5.62 + 2.10 × g hyp intake/d (r2 = 0.56; P < 0.001). Increasing oxalate intake from 13 to 93 mg/100 g DM did not affect Uox excretion but resulted in an increase in fecal oxalate output (P < 0.001) and positive oxalate balance (32.20 ± 2.06 mg/d). The results indicate that the intestinal absorption of the supplemental oxalate, and thereby its contribution to Uox, was low (5.90% ± 5.24%). Relevant increases in endogenous Uox excretion were achieved by increasing dietary hyp intake. The hyp-containing protein sources should be minimized in Ca oxalate urolith preventative diets until their effect on Uox excretion is tested. The oxalate content (up to 93 mg/100 g DM) in a diet with moderate Ca content does not contribute to Uox content.



INTRODUCTION

One of the most common uroliths detected in the urinary tract of cats is Ca oxalate, with an increasing prevalence over the past 30 yr (Picavet et al., 2007; Osborne et al., 2009). Urinary oxalate (Uox) in cats can originate from dietary components and from endogenous synthesis in the liver, with glyoxylate being the main precursor (Dijcker et al., 2011).

Studies in humans (Knight et al., 2006), rats (Ribaya and Gershoff, 1981, 1982; Bushinsky et al., 2002; Takayama et al., 2003; Ogawa et al., 2007), and mice (Jiang et al., 2012) have shown that substantial amounts of the glyoxylate and oxalate are synthesized endogenously through the metabolism of the AA hydroxyproline (hyp). In a randomized controlled trial, cats fed diets containing collagen (rich in hyp) had a 2- to 3-fold greater Uox excretion compared with diets containing soy isolate and horse meat as the protein source (Zentek and Schulz, 2004). However, it is not possible to ascribe this difference in oxalate excretion solely to the hyp content of the diets because other nutrients (particularly AA) also differed among the diets.

In humans, dietary oxalate intake contributes to the oxalate excreted in urine (Holmes et al., 2001; von Unruh et al., 2003). Intestinal absorption of dietary oxalate is highly dependent on the presence of dietary Ca, as Ca can form unabsorbable complexes with oxalate (Liebman and Chai, 1997; Holmes et al., 2001). Because feline diets have a greater Ca content (relative to oxalate) than human foods, it can be postulated that intestinal oxalate absorption in cats, and thereby its contribution to Uox, is lower than in humans. The study reported here aimed to determine the influence of dietary hyp as well as dietary oxalate (with moderate Ca intake) on urinary and fecal oxalate excretion in healthy adult cats.


MATERIALS AND METHODS

This study received prior approval from the Animal Ethics Committees of the Massey University (authorization number 2012/13), Wageningen University (authorization number 2011/113), and Royal Canin S.A.S. (authorization date February 27, 2012).

Animals and Housing

Eight domestic shorthair, intact female cats 1.5 to 8.5 yr of age (5.0 ± 0.4 yr) and weighing 2.7 to 3.4 kg (3.0 ± 0.0 kg) were used. Before initiation of the study, all cats underwent a veterinary examination (general physical exam, weighing, determination of BCS, and measuring urine pH and density) and were deemed healthy. The cats had been previously vaccinated with a live attenuated, freeze-dried vaccine containing feline panleucopenia, herpesvirus, and calicivirus (Fevac 3 Vaccine; Pfizer, New York, NY).

During d 1 to 5 of each feeding period, the cats were housed as a single group in a semioutdoor pen (4.5 × 1.4 × 2.5 m) at the Centre for Feline Nutrition (Massey University, Palmerston North, New Zealand). Twice daily (from 0830 to 0900 and 1500 to 1530 h) they were placed into individual metabolism cages (0.8 × 0.8 × 1.1 m) with a solid floor as described by Hendriks et al. (1999) and fed. During d 6 to 12, cats were housed individually in the metabolism cages for 23.5 h per day and were regrouped for 30 min for socialization. During these 30 min, cats were continuously observed to ensure that no urine or feces were voided. The trial was conducted from June to July 2012, and the mean maximum and minimum outdoor temperatures were 12.4°C ± 0.3°C and 4.7°C ± 0.5°C, respectively.

Study Design

The cats were fed 4 experimental diets formulated to contain equal amounts of ME, according to a Latin square design. Each diet was fed for a 12-d period, consisting of 7 d of adaptation (Dijcker et al., 2012) followed by 5 d of quantitative urine and feces collection.

Diets

Three dry extruded diet portions were formulated(The European Pet Food Industry Federation (FEDIAF), Brussels, Belgium, and The Association of American Feed Control Officials (AAFCO), Champaign, IL) to differ only in hyp content, i.e., high (Hhyp), moderate (Mhyp), and low hyp (Lhyp) diets. In the Hhyp and Mhyp diets, l-hyp (99% purity; Jinzhou JiHai Ltd., Jinzhou City, China) replaced brewer’s rice in the Lhyp diet to maintain a similar energy density. Two moist diet portions were formulated (AAFCO) to differ only in oxalate content, i.e., low (Lox) and high oxalate (Hox) diets. The ingredients of the diets are presented in Table 1. The moist diet portions were prepared once before the study by adding deionized water (Lox) or a sodium oxalate (May and Baker, Dagenham, Essex, UK) solution (Hox) into a commercial complete and balanced canned cat food (Heinz Wattie’s, Hasting, New Zealand) using a mixer (Hobart Manufacturing Co., London, UK). Daily portions of the moist diet were stored at –20°C and defrosted before feeding. Each experimental diet consisted of a mixture of 1 of the dry extruded diets and 1 of the moist diet portions. To obtain 4 experimental diets formulated differing only in hyp and oxalate content, the following combinations were made: 1) Hhyp and Lox (Hhyp-Lox), 2) Mhyp and Lox (Mhyp-Lox), 3) Lhyp and Lox (Lhyp-Lox) as a control, and 4) Lhyp and Hox (Lhyp-Hox). The dry extruded portions (Hhyp, Mhyp, and Lhyp) provided 94% of the daily maintenance energy requirement, and the moist diet portions (Lox and Hox) provided 6% of the daily maintenance energy requirement. The nutrient composition of these experimental diets was calculated from the analyzed nutrient composition of the dry (Hhyp, Mhyp, or Lhyp) and moist (Lox or Hox) diet portions used for the 4 combinations (Table 2). The oxalate content of the Hox diet was calculated from the analyzed value of the control diet and the added amount of oxalate. Food allowance was calculated according to an adult maintenance energy requirement of 418 kJ×kg−1 BW0.67×d−1 (NRC, 2006) and adjusted (if required) during the study to ensure BW maintenance. Body weight was recorded at d 1 and 7 of each feeding period, and feed refusals were determined daily. Fresh tap water was provided ad libitum and was replenished once a day.


View Full Table | Close Full ViewTable 1.

Ingredients of the dry and moist diets that formed the basis of the experimental diets

 
Diet portion1
Dry extruded Moist
Brewer’s rice Beef lung
Soy protein isolate hydrolysate Poultry offal (heart, liver etc.)
Fish meal Poultry by-products
Chicken fat Poultry waste
Powdered cellulose Starch (Avongold superfine)
Hydrolyzed poultry liver Carageenan gelling agent (ricogel)
Egg powder Vitamin and mineral premix2
Vegetable oil Sodium oxalate3
Fructo-oligosaccharides
Hydroxyproline4
Taurine
Vitamins and minerals5
Preservatives and antioxidants
1Each experimental diet was obtained by mixing a complete dry extruded diet portion (high, medium, or low hydroxyproline [Hhyp, hyp, or Lhyp]) with a complete moist diet portion (low or high oxalate [Lox or Hox]). The dry extruded portions provided 94% of the daily maintenance energy requirement (MER), and the moist diet portions provided 6% of the daily MER.
2Vitamin and mineral premix contained vitamin D3, vitamin E, thiamin, pantothenic acid, pyridoxine, folic acid, Fe, Zn, Mn, I, and Se.
3Only in the Hox moist diet portion.
4Only in the Hhyp and Mhyp dry extruded portions.
5Vitamin and mineral premix contained dl-alpha-tocopherol, inositol, niacin, l-ascorbyl-2-polyphosphate, d-calcium pantothenate, biotin, pyridoxine hydrochloride, riboflavin, thiamine mononitrate, vitamin A acetate, folic acid, vitamin B12 supplement, vitamin D3 supplement, choline chloride, calcium sulfate, calcium carbonate, sodium silico aluminate, potassium chloride, zinc, proteinate, zinc oxide, calcium iodate, and sodium selenite.

View Full Table | Close Full ViewTable 2.

Analyzed composition of diets1,2

 
Diet
Component, % Hhyp-Lox Mhyp-Lox Lhyp-Lox Lhyp-Hox
Hydroxyproline 3.83 1.96 0.20 0.20
Oxalate 0.012 0.016 0.013 0.093
DM (as fed) 72.19 72.37 72.09 72.30
CP 39.66 38.99 36.78 36.69
Crude fat 15.16 15.01 15.19 15.28
Ash 6.69 6.74 6.66 6.74
Total dietary fiber 5.22 5.47 4.87 4.85
Starch 31.44 32.68 30.88 30.79
Ca 0.96 0.94 0.96 0.97
P 0.92 0.89 0.95 0.96
Na 0.66 0.66 0.65 0.68
K 0.93 0.90 0.87 0.87
Asp 3.39 3.40 3.39 3.39
Thr 1.34 1.33 1.36 1.36
Ser 1.68 1.67 1.70 1.70
Glu 5.73 5.82 5.85 5.84
Gly 1.87 1.90 1.92 1.92
Ala 1.87 1.88 1.90 1.90
Val 1.76 1.76 1.69 1.69
Ile 1.48 1.50 1.46 1.46
Leu 2.70 2.70 2.70 2.69
Tyr 1.17 1.18 1.18 1.18
Phe 1.65 1.66 1.67 1.66
His 0.80 0.80 0.81 0.80
Lys 2.10 2.12 2.10 2.10
Arg 2.35 2.42 2.40 2.39
Pro 1.95 1.84 1.89 1.89
ME,3 MJ/100 g DM 1.89 1.88 1.89 1.89
1Low oxalate (Lox) with high hydroxyproline (hyp; Hhyp-Lox), moderate hyp (Mhyp-Lox), and low hyp (Lhyp-Lox) and low hyp with high oxalate (Hox; Lhyp-Hox) diets.
2On a DM basis unless indicated otherwise.
3Calculated according to NRC (2006).

Feces and Urine Sampling

The 5-d quantitative fecal and urine collection (d 8 to 12) was achieved using a modified litter box as described by Hendriks et al. (1999). Daily fecal samples collected on the top tray of the litter box were weighed, collected, and stored at –20°C. After freeze drying and grinding, fecal samples were analyzed for DM, Ca, and oxalate.

Urine samples were collected daily from the bottom tray of the litter box, which contained 5 mL of 3 N HCl to acidify the urine immediately after voiding for conservation. The weight of the urine/HCl mixture was determined by subtracting the weight of the bottom tray containing the urine mixture from the weight of the empty tray. The urine/HCl mixture from each cat was then added to a single pooled urine container, which was stored at –4°C. After 5 d of collection, the pooled urine samples were titrated to a pH between 1.5 to 2.0 with 3 N HCl (as was required for the analysis of oxalate, Ca, P, Mg, Na, K, ammonia, citrate, urate, sulfate, and creatinine), analyzed for specific density, and stored at –20°C. Specific density of the urine/HCl mixture was determined by comparing the weight of 1 mL of the urine/HCl mixture to 1 mL of distilled water. Daily urine volume was calculated by dividing the grams of collected urine by the specific density of the acidified urine, followed by the subtraction of the amount of HCl added to the urine. The acidified urine samples were analyzed for oxalate, Ca, P, Mg, Na, K, ammonia, citrate, urate, sulfate, and creatinine, and their concentrations were all corrected for the addition of HCl.

An additional nonacidified urine sample was collected from each cat overnight before the start of the 5-d quantitative urine collection on d 7. The procedure of urine sampling was as described before, except for the addition of 3 N HCl to the bottom tray of the litter box. Each sample was conserved by addition of 0.25 mL 20% chlorhexidine (Sigma-Aldrich, Gillingham, UK), stored at –4°C, and analyzed for pH and specific density after each feeding period. The pH of the urine was determined using a pH meter (MP220 pH Meter; Mettler Toledo, Columbus, OH).

Chemical Analyses

The diets were analyzed for DM and ash by drying to a constant weight at 103°C and combustion at 550°C, respectively. The CP (International Organization for Standardization [ISO], 2008), crude fat (ISO, 1999), total dietary fiber (AOAC, 1995), starch (Association Française de Normalisation, 1997), P (European Economic Committee, 1971), and Ca and Na (ISO, 2000) were determined, and the diets were analyzed for AA, including hyp, by cation exchange, HPLC separation, and ninhydrin-reactive colorimetric detection (Eppendorf-Biotronik LC 3000 Amino Acid Analyzer; Bioritech, Chamarande, France) following acid hydrolysis.

Oxalate, Ca, P, Mg, Na, K, ammonia, citrate, urate, sulfate, and creatinine were determined by ionic chromatography (Dionex, Port Melbourne, Australia; Royal Canin European Regional Lab, Aimargues, France) as described by Markwell et al. (1999). Oxalate in the feces and diets was determined by putting 3 to 5 g of product into 100 mL of an aqueous solution of HCl at 0.5% and boiling for 15 min. The solution was filtered, and the filtrate was centrifuged before dilution in water. The extract was analyzed by capillary zone electrophoresis, with reverse UV detection and quantification by internal calibration with sodium chlorate (InVivo labs, Saint-Nolff, France, personal communication).

Statistical Analysis

To test the influence of diet on urine characteristics and the balance of oxalate and Ca, analysis of covariance was performed using the mixed models procedure of SAS (version 9.2; SAS Inst. Inc., Cary, NC) with the covariates, diet, and period as fixed effects and cat as a random effect. Some of the dependent variables were log10 or 1/x transformed to attain normal distribution of the residuals. Within the model, differences among least squares means were compared with a Tukey-Kramer test only after a diet effect (P < 0.05) was observed. The dose-response equation for hyp intake on Uox excretion was determined by the univariate regression analysis using the REG procedure of SAS. Data were compiled into means ± SEM using SAS, and the level of significance was set at P < 0.05.


RESULTS

All cats remained healthy throughout the study and maintained their BW within 5% of the BW recorded at the start of the study. There was no difference in the energy intake of the cats among diets, and the values for the Hhyp-Lox, Mhyp-Lox, Lhyp-Lox, and Lhyp-Hox diets were 342.0 ± 14.4, 331.1 ± 9.3, 337.2 ± 9.6, and 341.1 ± 12.1 kJ·kg–1 BW·d–1, respectively.

There was an overall effect of diets on urine volume, ammonia, and sulfate (P = 0.017, 0.010, and 0.035, respectively; Table 3). Fecal quality remained good throughout the study, except for 4 cats having intermittent periods of softer feces. Urine volume and sulfate were lower in cats fed the Hhyp-Lox diet compared with the Lhyp-Lox diet (P < 0.05). Urine ammonia was greater in cats fed the Mhyp-Lox diet compared with the Lhyp-Hox diet (P < 0.005). Urinary pH, specific density, and the concentration of Ca, P, Mg, Na, K, citrate, urate, and creatinine were not different among diets.


View Full Table | Close Full ViewTable 3.

Mean values for selected urine characteristics in adult cats fed experimental diets1,2

 
Diet
P-value
Item Hhyp-Lox Mhyp-Lox Lhyp-Lox Lhyp-Hox
Volume, mL·cat–1·d–1 50.63 ± 3.40a 55.70 ± 1.96a,b 57.63 ± 1.68b 56.21 ± 2.55a,b 0.017
pH2 6.98 ± 0.21 7.34 ± 0.22 7.13 ± 0.16 6.86 ± 0.17 0.298
Specific density,3 g/mL 1.061 ± 0.002 1.060 ± 0.007 1.057 ± 0.004 1.056 ± 0.004 0.081
Oxalate, mmol/L 2.26 ± 0.17c 1.57 ± 0.07b 1.11 ± 0.08a 1.22 ± 0.07a <0.001
Ca,4 mmol/L 1.69 ± 0.48 2.03 ± 0.34 2.09 ± 0.68 3.24 ± 1.04 0.194
P, mmol/L 71.28 ± 3.43 76.50 ± 2.65 76.26 ± 3.26 78.04 ± 2.72 0.050
Mg, mmol/L 2.71 ± 0.30 3.16 ± 0.41 2.85 ± 0.28 2.94 ± 0.35 0.189
Na, mmol/L 241.6 ± 4.3 235.7 ± 8.6 240.3 ± 7.6 243.1 ± 9.3 0.808
K, mmol/L 190.0 ± 5.6 195.0 ± 4.7 195.0 ± 5.4 188.8 ± 5.0 0.113
Ammonia, mmol/L 200.0 ± 5.2a,b 205.7 ± 5.1b 199.7 ± 7.1a,b 191.3 ± 7.6a 0.010
Citrate,4,5 mmol/L 2.85 ± 0.90 1.70 ± 0.31 1.70 ± 0.44 1.69 ± 0.44 0.072
Urate, mmol/L 1.25 ± 0.11 1.20 ± 0.11 1.18 ± 0.08 1.16 ± 0.09 0.122
Sulfate, mmol/L 113.3 ± 2.3a 116.1 ± 2.7a,b 119.49 ± 3.8b 115.6 ± 3.3a,b 0.035
Creatinine, mmol/L 25.56 ± 0.97 26.11 ± 0.78 25.84 ± 0.58 24.76 ± 0.78 0.114
a–cWithin a row, means without a common superscript letter differ (P < 0.05).
1Low oxalate (Lox) with high hydroxyproline (hyp; Hhyp-Lox), moderate hyp (Mhyp-Lox), and low hyp (Lhyp-Lox) and low hyp with high oxalate (Hox; Lhyp-Hox) diets.
2Values are means ± SEM (n = 8).
3Measured in nonacidified urine. Number of animals: Hhyp-Lox and Mhyp-Lox, n = 7; Lhyp-Lox, n = 6; and Lhyp-Hox, n = 5.
4Urinary Ca and citrate concentrations were log10 transformed for statistical analysis.
5After excluding 1 high citrate value of the Hhyp-Lox diet from the analysis, diet was no longer a significant factor (P = 0.072).

Increasing hyp in the diet resulted in greater mean Uox concentrations (Table 3) and daily urinary oxalate excretion (Table 4; Lhyp-Lox vs. Mhyp-Lox vs. Hhyp-Lox; P < 0.05). A statistically significant (P < 0.001; r2 = 0.562) linear dose response was observed between the hyp intake (g·cat–1·d–1) and Uox (mg·cat–1·d–1): y = 5.622 + 2.097x (SE is 0.536 and 0.395 for intercept and regression coefficient, respectively; P < 0.001).


View Full Table | Close Full ViewTable 4.

Oxalate and Ca balance in adult cats fed experimental diet1,2

 
Diet P-value
Item Hhyp-Lox Mhyp-Lox Lhyp-Lox Lhyp-Hox
Oxalate,3 mg·cat–1·d–1
    Intake 6.55 ± 0.12a 8.56 ± 0.06c 7.26 ± 0.05b 50.83 ± 0.80d <0.001
    Fecal output 5.19 ± 0.43a 5.41 ± 0.25a 5.13 ± 0.35a 12.52 ± 2.34b <0.001
    Urinary output 10.12 ± 0.80c 7.83 ± 0.25b 5.75 ± 0.39a 6.10 ± 0.17a <0.001
    Balance –8.76 ± 0.74b –4.68 ± 0.41a –3.61 ± 0.45a 32.20 ± 2.06c <0.001
Calcium, mg·cat–1·d–1
    Intake 521.3 ± 9.4a,b 507.4 ± 3.5a 516.6 ± 3.6a,b 528.9 ± 9.3b 0.011
    Fecal output 564.3 ± 46.7 534.9 ± 27.2 513.4 ± 20.5 547.4 ± 47.3 0.709
    Urinary output 3.18 ± 0.78 4.41 ± 0.66 4.81 ± 1.60 7.40 ± 2.46 0.112
    Balance –46.20 ± 39.61 –31.97 ± 25.35 –1.61 ± 21.75 –25.89 ± 42.05 0.497
a–dWithin a row, means without a common superscript letter differ (P < 0.05).
1Low oxalate (Lox) with high hydroxyproline (hyp; Hhyp-Lox), moderate hyp (Mhyp-Lox), and low hyp (Lhyp-Lox) and low hyp with high oxalate (Hox; Lhyp-Hox) diets.
2Values are means ± SEM (n = 8).
3Oxalate intake and urinary oxalate and calcium output were log10 transformed. Fecal oxalate and calcium output and oxalate balance were 1/x transformed.

Fecal oxalate and Ca output were not different among the 3 Lox diets (Table 4). The calculated oxalate balance of cats was more negative (P < 0.05) for the cats on the Hhyp-Lox compared with the Mhyp-Lox and L-hyp-Lox diets and tended to be different (P = 0.084) between the Mhyp-Lox and the Lhyp-Lox diets. Although Ca intake (mg·cat–1·d–1) tended to differ when feeding the Mhyp-Lox and Lhyp-Hox diets (P = 0.008), the Ca balance was not different among diets.

Urinary oxalate and all other urine characteristics remained unaffected with increasing oxalate intake (Lhyp-Lox vs. Lhyp-Hox; Table 3). Fecal oxalate output and the calculated oxalate balance of the cats were increased (P < 0.05) when they were fed the LHyp-Hox compared with the Lhyp-Lox diet (Table 4). The fecal oxalate output for cats on the LHyp-Hox diet was highly variable (0% to +450% compared with the control diet). On the basis of the differences in Uox excretion of the cats when the Lhyp-Lox and Lhyp-Hox diets were fed, it was calculated that 5.90% ± 5.24% of the Uox excretion was due to absorption of the supplemented oxalate (assuming that 100% of the difference in Uox excretion among these diets was due to absorption of the supplemented oxalate), which accounted for 0.78% ± 0.78% of the total amount of supplemented oxalate.

The relative increase in Uox excretion in cats fed the Hhyp-Lox, Mhyp-Lox, and Lhyp-Hox diets compared with the unsupplemented control diet (Lhyp-Lox) is shown in Fig. 1. Compared with the control diet, the Uox excretion increased by 78.5% ± 14.6% and 39.8% ± 8.6% with the Hhyp-Lox and Mhyp-Lox diets, respectively. It increased by 8.6% ± 6.0% with the Lhyp-Hox diet.

Figure 1.
Figure 1.

Box plots of the increase in urinary oxalate excretion in adult cats fed low oxalate (Lox) with high hydroxyproline (hyp; Hhyp-Lox), moderate hyp (Mhyp-Lox), and low hyp (Lhyp-Lox, control) and low hyp and high oxalate (Lhyp-Hox) diets relative to the urinary oxalate excretion when fed the control diet. The box represents the values within the 25th and 75th percentiles, and the whiskers are the minimum and maximum values. The numbers represent means.

 

DISCUSSION

The present study showed that dietary hyp increases endogenous Uox excretion in healthy adult cats. The additional Uox excretion with increasing hyp intake (Lhyp-Lox, Mhyp-Lox, and Hhyp-Lox) was of endogenous origin because the fecal oxalate output was similar among these diets, and the greater negative oxalate balance indicated that endogenous synthesis of oxalate occurred. The l-hydroxyproline can be metabolized to glyoxylate and then to oxalate in the hepatocyte (Ribaya and Gershoff, 1981, 1982; Bushinsky et al., 2002; Takayama et al., 2003; Ogawa et al., 2007; Jiang et al., 2012) and subsequently excreted in the urine. In the present study, 0.32% (on a molar basis) of the supplemented hyp was recovered as oxalate in the urine. To the authors’ knowledge, no data are available to calculated dietary oxalate recoveries in cats or other animal species. In a human study where hyp from gelatin was fed, approximately 0.5% was recovered (Knight et al., 2006). This fairly similar recovery rate between cats and humans is unexpected, as cats have their alanine:glyoxylate aminotransferase 1 (AGT1; catalyzing the conversion of glyoxylate to Gly) mainly located in the mitochondria (Danpure et al., 1990; Lumb et al., 1994), whereas in humans, the majority of AGT1 is located in the peroxisomes (Danpure, 2006). The AGT1 located in mitochondria mainly converts AA-derived glyoxylate, whereas AGT1 in peroxisomes mainly converts sugar-derived glyoxylate. However, as the digestibility of both hyp sources (i.e., close to 100% for synthetic hyp and 82% for gelatin; Laser Reutersward et al., 1985) was likely to be different, cats may be more efficient in preventing oxalate formation from hyp (via glyoxylate) than humans.

It is likely that hyp present in animal protein sources commonly used to manufacture pet food has a lower intestinal absorption compared with the synthetic hyp tested in the present study. The hyp in collagen tissue from melted fat is also likely to be highly available for intestinal absorption and, as a consequence, also for endogenous oxalate synthesis. This may be concluded from the study by Zentek and Schulz (2004), where Uox excretion was increased after feeding cats diets containing collagen tissue (from melted fat) as the protein source compared with diets containing horse meat and soya isolate as the protein source. However, the effect of other hyp-containing protein sources (such as fish and poultry meal) on endogenous oxalate excretion in cats remains to be determined.

It seems unlikely that net intestinal secretion of endogenous oxalate occurred because the increase in urinary (endogenous) oxalate excretion with increasing hyp intake was not accompanied by an increased fecal oxalate output. This finding was unexpected because oxalate was found in fecal samples after feeding rats an oxalate-free diet (Ribaya and Gershoff, 1982), indicating that endogenously synthesized oxalates are secreted into the rat intestinal tract. Although the potential interference of oxalate-degrading bacteria in the intestine cannot be excluded, the intestinal oxalate output was constant despite increased endogenous oxalate synthesis from hyp intake. It seems therefore unlikely that endogenous Uox excretion can be reduced by promoting intestinal secretion of endogenous oxalate in healthy cats.

Dietary oxalate supplementation did not increase Uox excretion. This was unexpected because in a recently conducted retrospective cohort study in cats, an estimated oxalate absorption of 6.2% was found (J. C. Dijcker et al., unpublished data). In the present study, it was estimated that 0.78% of the added dietary oxalates was found in the urine. An explanation may be the relatively high Ca concentration of feline diets, which may have resulted in increased oxalate binding in the intestinal lumen, thereby making oxalate less available for absorption. In the present study, the diets contained a moderate Ca concentration (0.51 in the Hhyp-Lox, Mhyp-Lox, and Lhyp-Lox diets and 0.52 g/MJ ME in the Lhyp-Hox diet). In a study with dogs fed diets containing various concentrations of Ca and oxalate, the low-Ca diets (0.43 g Ca/MJ ME and 24, 42, and 60 mg oxalate/MJ ME) resulted in an inconsistent increase in Uox excretion (Stevenson et al., 2003). In a recent retrospective cohort study (J. C. Dijcker et al., unpublished data), no obvious relationship was found among the dietary Ca to oxalate ratio and Uox excretion in cats. However, the effect of a low Ca concentration on Uox excretion in cats remains to be tested in a prospective setting.

Because dietary supplementation with oxalate (with a moderate Ca content) did not affect Uox excretion, intestinal oxalate absorption (estimated at 0.78% ± 0.78%) and its contribution to Uox (5.90% ± 5.24%) may be considered to be low. However, these characteristics may have been underestimated as the reference diet was not oxalate free and contained a small amount of oxalate. A more accurate estimation may be provided by the use of an oxalate-free diet as the reference diet, which is difficult to produce because of the constraints on extrusion of solely oxalate-free ingredients.

It can be argued that supplementation of diets with oxalate up to 49 mg/MJ ME (Lhyp-Hox diet) may not have been sufficient to increase oxalate absorption and thereby Uox excretion. However, the dietary oxalate content of 255 commercial feline diets was recently reported to be 27.4 ± 0.4 mg/MJ ME with a range of 2 to 118 mg/MJ ME (J. C. Dijcker et al., unpublished data). The oxalate content of the Lhyp-Hox diet falls within the upper 10% of this range. The Na oxalate used in the present study has been shown in humans to have a similar (Prenen et al., 1984) or greater (Brinkley et al., 1981; Hanson et al., 1989) digestibility than oxalate present in food items such as rhubarb and spinach. Although it is unknown whether the digestibility of Na oxalate and oxalate in feline diets is comparable, the absence of an effect of Na oxalate on Uox excretion in the present study indicates that the oxalate content of commercial feline diets does not appear to be an important causative factor affecting Uox excretion. As such, dietary oxalate seems unlikely to be a major contributing factor to the prevalence of Ca oxalate uroliths in cats.

Absorbed or endogenously synthesized oxalate is quantitatively excreted and not stored in the body (Curtin and King, 1955). The positive oxalate balance of 32.20 ± 2.06 mg·cat·d–1 following the consumption of the Lhyp-Hox diet therefore indicates that oxalate were degraded in the intestinal tract or metabolized to other components. It is likely that oxalate was used as a substrate by oxalate-degrading bacteria in the intestines. In several in vitro studies with feline and canine fecal samples, oxalate-degrading bacteria species have been identified (Weese et al., 2004, 2009;Murphy et al., 2009; Weese and Palmer, 2009; Ren et al., 2011; Gnanandarajah et al., 2012). Future feline studies feeding diets with different oxalate concentrations (balanced for Ca) followed by identification of fecal oxalate-degrading activity may provide more information. Whether Uox excretion can be reduced by promoting intestinal oxalate-degrading bacteria, however, needs to be studied.

In the present study, the Ca balance was measured to monitor a known dietary factor potentially affecting intestinal oxalate absorption and also to provide more information on the accuracy of intake and output measurements. The mean Ca balance recorded in the present study of –8.2% to –0.3% (relative to the Ca intake) is similar to Ca balances (of –7.1% to +8.6%) observed in another study with adult cats fed comparable amounts of Ca (Pastoor et al., 1994). In addition, the Ca balance in the present study did not differ among diets.

In the present study, dietary hyp, rather than dietary oxalate, increased Uox excretion in adult cats. The finding that dietary hyp induces endogenous Uox excretion indicates that the use of hyp-containing protein sources should be minimized in Ca oxalate urolith preventative diets until their effect on Uox excretion is determined. The increasing endogenous Uox excretion was not accompanied by increased fecal oxalate excretion, making net intestinal secretion of endogenous oxalate with the present conditions (moderate Ca content diet) unlikely. Dietary oxalate supplementation to a diet with a moderate Ca content did not affect Uox excretion in this study. Therefore, it may be concluded that intestinal oxalate absorption does not appear to contribute to Uox excretion and as such to the prevalence of Ca oxalate urolithiasis in cats. The disappearance of intestinal oxalate after feeding a high-oxalate diet found in the present study may be explained by the presence of oxalate-degrading bacteria.

 

References

Footnotes


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