Based on the amount and source of dietary phosphate as well as time of exposure, phosphate containing food and feed additives, i.e. inorganic phosphates, can cause adverse health effects, especially on kidney function, in humans 123 and animals 45678910. Sodium monophosphate (NaH₂PO₄) supplementation caused a decrease in creatinine clearance and increased phosphaturia in cats ^{6, 11}. In pigs, inorganic phosphate intake resulted in tubular calcification, inflammation, fibrosis, glomerular degeneration and atrophy 12 while in dogs, this led to a disruption of calcium and phosphate homeostasis including an increase of parathyroid hormone (PTH) and fibroblast-growth-factor 23 (FGF23) 7. The intensity of these effects are influenced by the solubility and therefore availability of the phosphate source 71314,which then effects phosphate homeostasis and renal phosphate excretion. As demonstrated in rodents, the amount of excreted phosphorus correlates directly with renal damage 15, for which reason the dietary supply is of utmost importance, especially because phosphate containing additives are widely used in processed pet food due to their diverse properties (e.g., water binding, preservation, palatability enhancement, dental calculus prophylaxis, and uroliths prevention via urine pH adjustment 1617181920). Additional factors influencing the effects of added inorganic phosphate on the body have to be taken into account: high calcium concentrations and a wide calcium to phosphorus ratio (Ca/P ratio), for example, are expected to lower the apparent digestibility of phosphate and vice versa 62122. However, as demonstrated in dogs, this correlation does not seem to apply to the same extend to highly soluble phosphate sources 23. Further major minerals including their electric charge are also of interest in this context. They influence the bodies’ cation anion balance (CAB), mineral balances 2425, and urine pH 242526272829. Various medical reasons require modification of dietary CAB in order to influence blood and urine pH 2430.

The aim of this study was therefore to examine the influence of different dietary CAB on the apparent digestibility and renal excretion of phosphate as well as on further parameters of phosphate homeostasis in cats fed additional inorganic phosphate (NaH₂PO₄).

Eleven healthy adult European shorthair cats (4 males, 7 females, 1-4 years of age, 2.7-4.7 kg body weight), bred and housed in the cattery of the chair of Animal Nutrition and Dietetics, Department of Veterinary Sciences, LMU Munich, participated in a total of three feeding trials (control group, CON; negative CAB, nCAB; positive CAB, pCAB). Each cat underwent a general health check including complete blood and measurement of selected parameters of kidney function (urea, creatinine, SDMA, serum electrolytes, urinalysis) before commencing the study. The diet period of 28 days (d) consisted of an adaption (18 d) and a digestibility phase (10 d). During the digestibility phase, urine and faecal samples were collected quantitatively and food and water intake were measured. Blood samples were drawn on day 28 preprandially (at least 12 hours (h) fasted) and postprandially (3 h after food intake). Cats were examined visually on a daily basis by a veterinarian and underwent a weekly general clinical exam and weighing. Proceedings and protocols were in alignment with the guidelines of the Protection of Animals Act and approved by the representative of the Veterinary Faculty for animal welfare, as well as the Government of Upper Bavaria (reference number ROB 55.2.-2532.Vet_02-19-38).

All cats remained clinically healthy throughout the study. Water intake and urine volume did not differ between groups. DM and ME intake decreased in the high phosphate (CAB) diets (nCAB: 10 ± 1; pCAB: 11 ± 1 g/kg BW/d) compared to CON (13 ± 1 g/kg BW/d; p > 0.001 and 0.003) and along with it, faecal excretion of DM (1.3 ± 0.3 vs. 2.0 ± 0.3 vs. 2.0 ± 0.3 g/mg/kg BW; p < 0.001). Apparent digestibility of DM did not differ between groups (CON: 90 ± 2; nCAB: 92 ± 1; pCAB: 91 ± 2 %). Apparent digestibility of phosphorus was significantly lower in diet nCAB compared to CON and pCAB (p < 0.001 and p = 0.017; Table 2). Compared to diet pCAB, the apparently digested amount of phosphorus was lower in diet CON (p < 0.001) and diet nCAB (p = 0.019) (Table 2). The phosphate retention was also significantly higher in diet pCAB compared to nCAB (p = 0.015).

The apparently digested (CON: p < 0.001; nCAB: p = 0.019) and retained (CON: p = 0.04; nCAB: p = 0.015) amount of phosphorus was highest in the pCAB group (Table 2). Irrespective of the CAB, urine phosphorus concentrations ((g/l); Table 4) as well as renal phosphorus excretion (mg/kg BW) was higher in both CAB groups compared to CON (p < 0.001). Even though the apparent calcium digestibility was not statistically different (p = 0.08), the apparently digested calcium was significantly reduced in diet pCAB (p = 0.04). In this group, the calcium retention was negative and significantly lower compared to CON (p = 0.04) while renal excretion of calcium did not differ between groups (p = 0.31; Table 2).

To date, knowledge about the effects of source and amount of other dietary minerals, or the combination thereof, on the consequences of inorganic phosphate intake is limited. Considering the exceptionally high prevalence of CKD in felines 35 and the established effects of inorganic phosphate intake on renal health, the aim of this research was to investigate the dietary CAB as a potential factor of influence on availability and selected effects of dietary inorganic phosphate. This is especially important in feline nutrition due to the variability of the CAB in cat food, for example in diets intended to influence urinary pH and therefore urolith formation. As struvite uroliths are soluble in acidic solutions 36, commercial diets commonly aim for a slightly acidic to neutral urine pH to prevent struvite formation 263738, while medical diets for struvite dissolution aim for a urine pH between 5.9 - 6.4 29. Because urine pH and CAB of a diet correlate closely, calculating the CAB allows the prediction of the average daily urine pH which in turn can be altered by adjusting the concentration of minerals in a diet 30. A CAB between -60 and +100 mmol/kg DM approximately results in urine pH values between 6.0-7.0 39. Accordingly, diets with a CAB ≤ 0 mmol/kg DM have been proven effective in the treatment and prevention of frequently occurring uroliths in dogs and cats and are therefore often prescribed 4041. The CAB of diet pCAB, however, results in the relatively high predicted urine pH value of 7.7.

In the current investigation, which was done applying a well-established study design 7811134243, the effects of feeding diets with added inorganic phosphate of an identical source and the same Ca/P ratio but different CAB due to the use of varying calcium sources (CaCO₃, CaCl₂) and methionine addition were investigated in a short term study. Calcium carbonate has an alkalizing effect while calcium chloride reduces CAB and urine pH 30. The use of these two calcium sources was necessary to adjust the CAB in the test diets with the lowest possible effect on the concentrations of other minerals. Whether the calcium source itself had a separate effect on the parameters measured in this study cannot be determined based on the current results. In addition, this research was conducted in a group of relatively young cats. Younger individuals were purposely selected to reduce the likelihood of early-stage kidney disease despite normal blood test results. Because energy and nutrients were apportioned based on the individual metabolic body weight of each cat, the nutrient supply per unit of body weight was consistent. Attention was also paid to a balanced gender distribution and an ideal body condition. Hence, the chosen test group is representative of a healthy adult cat population.

Adding inorganic phosphate in the form of sodium phosphate to a balanced control diet containing solely organic phosphates caused a significantly altered phosphate balance in both test diets. Beyond this, a positive CAB of + 450 mmol/kg DM led to a significantly higher apparent digestibility and retention of phosphate: compared to the diet nCAB with a negative CAB of -10 mmol/kg DM, the amount of apparently digested phosphate per kg BW was about 50 % higher and differed also significantly from CON. These results are supported by previous research: acidification of the diet in a study by Ching et al. (1989) also led to lower apparently digested phosphate and reduced phosphate retention 24. Similarly, Pastoor et al. (1994) observed lower urinary phosphate concentrations when adding CaCl₂ compared to CaCO₃ to the diet of healthy cats 44.

A possible explanation is the existence of chemical interactions between different mineral compounds in the feed. In the present study, NaH₂PO₄ was used in combination with either CaCO₃ alone or with a mixture of CaCO₃ and CaCl₂, respectively. In aqueous solutions, these compounds can react as follows:

nCAB: NaH₂PO₄ + CaCl₂→ Ca(H₂PO₄) + NaCl

pCAB: NaH₂PO₄ + CaCO₃→ Ca₃(PO₄) + H₂O + 3 CO₂ + 2 NaOH

In diet nCAB, the described reaction results in calcium hydrogen phosphate (DCPA, Ca(H₂PO₄)) formation, while in diet pCAB it is more likely that tricalcium phosphate (TCP, Ca₃(PO₄)) is formed. In acidic solutions, such as those found in the cat's stomach, TCP exhibits a considerably higher solubility than DCPA 4546. As solubility and bioavailability of minerals are closely connected, the higher solubility of TCP could explain the increase of apparently digested phosphate in diet pCAB, irrespective of the diet’s CAB.

In alignment with the amount of apparently digested phosphate, the postprandial serum phosphate values increased in group pCAB and were significantly higher than in CON, exceeding the reference range in 5/11 cats. Consequently, the postprandial sCaxP rose above the threshold of 55 mg²/dl² introduced by Block et al. (2000) in 9/11 cats 34. An increased sCaxP should be avoided because it increases the risk of soft tissue calcification 47 and is negatively correlated with life expectancy for example in human and canine kidney patients 4849.

Serum FGF23 values, an early marker of CKD, were significantly increased pre- and postprandially in group pCAB, but not in group nCAB despite the same supply with inorganic phosphate (NaH₂PO₄+2H₂O), causing values above the threshold of 300 pg/ml in 3/11 cats. Presumably, this was caused by the higher amount of apparently digested phosphate and the increased serum phosphate concentration in this group. As a phosphatonin, FGF23 increases renal phosphate excretion in response to elevated serum phosphate concentrations, thereby regulating phosphate homeostasis 5051. Apart from the direct connection between renal health and the amount of phosphate excreted per nephron 15, increased FGF23 serum values were shown to have additional direct adverse health effects, such as the disruption of vitamin D and bone metabolism 5253 or cardiovascular dysfunction 5455. Despite the increased serum FGF23 concentrations in pCAB, the only numerically higher urinary P/Crea ratio 56 (Table 4) and absolute amount of urinary phosphate excretion per kg BW in group pCAB compared to nCAB indicate that renal phosphate excretion was only slightly affected. The lower urinary creatinine values in the CAB diets can be explained by the overall lower food intake compared to CON.

Digestibility, availability and consequently potential adverse health effects of inorganic phosphates do not solely depend on their amount and source, but also on the supply with other minerals such as sodium 42. In this study, significant effects of sodium monophosphate as a source of inorganic phosphate on the phosphate homeostasis were determined only in the diet with a relatively high CAB. However, previous research suggests that diets with a negative CAB might affect health for different reasons, regardless of the phosphate consumption. Several studies in cats 242529 demonstrated adverse effects such as metabolic acidosis and alterations of the animal’s mineral balance after ingestion of acidifying diets. Consequently, reducing the dietary CAB is not a viable solution to attenuate possible adverse health effects of highly soluble inorganic phosphates added to the diet of cats.

In this study, a dietary CAB of 450 mmol/kg dry DM in a diet containing sodium monophosphate led to a significant increase of apparently digested phosphate, phosphate retention, serum phosphate and serum FGF23. Consequently, potential health risks due to the intake of inorganic phosphates can only be evaluated when extensive information about the composition and ingredients of a diet are considered. Concluding from the results of the present study, additional research regarding possible effects of phosphate containing food additives in combination with other dietary factors is required before postulating a safe upper limit, guaranteeing that inorganic phosphate is unconditionally safe for human and animal consumption.