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.