1. Introduction
The majority of body phosphorus (P) can be found in bones and teeth, where it forms hydroxyapatite crystals with calcium (Ca) [
1,
2]. Subsequently, inadequate dietary P intake can negatively impact bone integrity and contribute to diseases like osteoporosis, rickets, or osteomalacia [
3,
4]. Aside from its function related to the skeleton, phosphates are involved in a plethora of cellular processes, such as enzyme activity, cell signaling, or blood buffering. The most important organs involved in the P homeostasis regulating P absorption, excretion, and storage are the gastrointestinal tract, kidney, and bone [
1,
2,
5]. Phosphorus and Ca homeostasis are closely linked as they are regulated by the same hormones, including vitamin D (VitD), parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23) [
6,
7,
8,
9].
Studies in mice and humans showed that the phosphaturic hormone FGF23 is primarily involved in P homeostasis and is produced by osteoblasts and osteocytes in the bone as well as by pericyte-like cells in the bone marrow as a response to high blood P and mainly suppresses P reabsorption and VitD activation in the kidney [
10,
11,
12]. Moreover, there is evidence from studies in mice, chickens, and humans that FGF23 is expressed in other tissues, including the stomach, small intestines, and lymph nodes [
11,
13,
14]. However, little is known of whether serum levels of the aforementioned regulatory factors correspond to intestinal Ca and P absorption as well as to bone and renal metabolism, especially in young growing adolescents. Moreover, there is evidence from studies in rodents, cows, and pigs that the transcellular and paracellular uptake in the intestine of Ca and P are linked and that the Ca and P availability and their ratio in the diet and digesta influence their reciprocal intestinal uptake [
15,
16,
17]. It was shown that enhanced intestinal availability and absorption of P negatively impact Ca metabolism and if leading to an P overload also negatively impact P metabolism. This is in particular the case when calcium intake is low to moderate as it is the case with phosphate-rich Western-style diets [
18,
19,
20,
21].
With regards to dietary sources, cereals and legumes are important sources of P for omnivore and herbivore species as well as for humans. A shift in human nutrition towards a healthier diet replacing meat with cereals and legumes is widely discussed to improve the overall nutritional quality of the diet. However, the majority of P in cereals is bound in the form of phytate-P, which has low digestibility in humans and monogastric animals because the gastrointestinal tract does not express the respective enzymatic capabilities for phytate hydrolysis [
22,
23,
24,
25]. By contrast, phytases are expressed by intestinal microbes, which renders phytate-bound P more available to the host, especially in species that rely on alloenzymatic digestion [
26,
27,
28]. In addition, the dietary Ca and P availability modifies the microbiota composition along the digestive tract [
29,
30]. In order to improve the phytate-P availability and to avert its potential negative impact on the intestinal availability of other minerals like Ca and protein through the formation of complexes with phytate, different food processing techniques have been applied. Traditional food processing methods used in human nutrition, which are also applied in animal nutrition, like soaking, malting, and fermenting of cereal grains, activate endogenous phytase in the germ [
23,
31,
32]. For instance, this technique is applied in bread making using sourdough fermentation or beer brewing or soaking of sorghum [
33,
34,
35]. An adaptation of these methods to make the phytate-degrading effect more controllable is the soaking of grains in mild organic acid solutions, such as lactic acid (LA). In using this method, we recently showed that the phytate-P content of maize, barley, and wheat of 2.2, 2.5, and 3.2 g/kg dry matter pre-incubation, respectively, decreased by 24.4% to 31.4%, which was accompanied by a similar increase in lower
myo-inositol phosphates. Aside from phytate-P, the soaking of cereal grains in LA also altered the content of other nutrient fractions in the grains, including cations (e.g., Ca), starch, hemicellulose, and protein [
36,
37]. Whether LA soaking of cereals, however, affects the intestinal, renal, and bone metabolism and/or improves mineral retention has not been sufficiently elucidated yet. Since imbalances in the dietary supply and availability affects the intestinal absorption and body metabolism of Ca and P, we hypothesized that an increased intestinal phytate-P and Ca release due to the LA treatment of cereals may enhance the apparent absorption and retention of Ca and P. We further hypothesized that the changes in absorption and retention of Ca and P should be reflected in increased bone formation, reduced renal excretion of Ca and P, as well as in the serum profile of regulatory hormones.
In order to test our hypothesis, we aimed to create different amounts of intestinally absorbable and subsequently systemically available P, either by soaking cereal grains in a 2.5% LA solution prior to feeding and/or by adding microbial phytase to the diet [
23,
38,
39]. Although phytase is not typically used in human food preparations, by adding phytase to our diets, we could achieve different intestinal and body availabilities of P without changing the total P content of our diets. In fact, the total P content was relatively similar across diets, which minimized the interference from different dietary P intakes on the intestinal and body Ca and P homeostasis. By using a 2 × 2 factorial design, we investigated the effects of phytase supplementation and LA treatment of cereals on the intestinal uptake and retention of Ca and P; intestinal, renal, and bone gene expression related to Ca and P homeostasis; bone parameters; as well as on the serum levels of regulatory hormones in growing pigs. We used the pig as a model as its digestive physiology and mineral metabolism are relatively similar to humans [
40,
41]. Since more evidence is available for adults [
42,
43,
44], we were especially interested in the role of FGF23 as a regulatory factor for P homeostasis in the growing phase.
4. Discussion
In the present study, we used phytase supplementation and LA treatment of cereals to create different amounts of intestinally absorbable and hence systemically available P to study their effect on parameters related to Ca and P metabolism in the intestine, kidney, and bone and their regulatory hormones in a growing pig model. Both, the phytase supplementation and the LA-treated cereal grains, improved the intestinal availability of P as indicated by the higher P solubility in gastric digesta as well as by increased apparent absorption and retention of P. Non-surprisingly, the effect on P absorption and retention was stronger with the phytase supplementation than with the LA-treated cereals. With the increased intestinal and subsequently systemic availability of P with both dietary treatments, we would have expected to observe an anabolic response of regulatory factors related to Ca, P, and bone metabolism along the intestinal mucosa as well as in the serum, kidney, and bones, especially in the young growing pigs. However, serum Ca, P, and hormones as well as renal and bone tissues showed different responses to the dietary treatments compared to those observed at the intestinal level. Soluble P fraction in gastric digesta may hint at diverging Ca to available P ratios in the gastrointestinal tract caused by the single and combined dietary treatments. This may have led to an unbalanced systemic availability of Ca and P due to the dietary treatments, lowering the effective utilization of Ca and P for osteogenesis and increasing their renal excretion. It is known that high intestinal availability and absorption of dietary phosphates can impair Ca homeostasis [
67,
68]. This assumption would be supported by the ratios of Ca: P calculated for serum, urine, and retention, which were drastically changed by phytase addition and the LA-treated cereals. Notably, physiological effects in the various organs often differed, depending on whether treatments were applied as a single treatment or combined.
Whilst the phytase supplementation mainly raised the dietary P, Ca, and amino acid (protein) availability according to the absorption data, the LA treatment of cereal grains additionally modified the dietary carbohydrate fractions, including the starch and hemicellulose fractions in the wheat and maize grains [
37]. LA treatment-related changes in the nutrient composition can be linked to the leaching of these nutrients (e.g., minerals and starch) into the soaking solution, activation of enzymes in the germ (e.g., hemicellulose), and acid hydrolysis (e.g., phytate-P) [
36,
37]. These changes likely contributed to the rise in the caecal concentration of total SCFA. Increased large intestinal fermentation and SCFA production has been reported to stimulate intestinal cation (e.g., Ca and Mg) absorption. The SCFA lower the luminal pH, which increases mineral solubility [
69], thereby increasing the mineral gradient between the luminal and serosal side, and allowing passive mineral transport to increase [
70]. This can be explained by the fact that Ca forms insoluble Ca-phosphate complexes at pH above 7 in the intestinal lumen [
71,
72]. Although we did not find a direct effect of the LA-treated cereals on caecal pH, the increased caecal SCFA concentrations with the LA-treated cereals may point in the direction that fermentation may have contributed to the overall mineral absorption in pigs fed the respective diets.
Under normal physiological conditions, the majority of the absorbed Ca and P should be used for osteogenesis in a fast-growing animal. With the applied feed allowances, pigs gained 600 g/day during the 19-day experimental period, which corresponded to the normal growth rate of a cross-bred meat-type pig of this age [
73]. Therefore, if the intestinally absorbed Ca and P led to a systemic overload, the excessive Ca and P should have been excreted via the kidneys as it has been shown for pigs [
74,
75,
76]. Noteworthy, in other animal species, the main regulation routes can differ, like dogs and cats, in which the Ca and P metabolism relies more strongly on regulatory mechanisms in the intestine and bone rather than in the kidney [
77,
78].
In general, Ca and P are mostly actively taken up via transporter-mediated transcellular pathways in the small intestine of pigs. Nevertheless, passive absorption via the paracellular route is relevant, especially with the increasing intestinal mineral availability shown in pigs and rats [
16,
79,
80]. In the present study, intestinal expression of Ca and P carrier proteins and signaling likely represented the combined effects of a local adjustment to the actual Ca and P concentrations in the respective intestinal segment as well as regulatory actions to adjust systemic availability of Ca and P due to VitD signaling. From the expression levels, the results supported that the main sites of active carrier protein-mediated transport of Ca and P were the duodenum and jejunum. Despite the improved intestinal P availability, the phytase supplementation did not alter the expression levels of the phosphate transporters
SLC34A1 and
SLC34A2. This may suggest that if the mRNA was translated into functional protein, the phytase supplementation did not modify transcellular P uptake, which potentially explains the increased serum P with the phytase-containing diet. Oppositely, the LA-treated cereals seemed to enhance the small intestinal P absorption, as indicated by the trends for higher
SLC34A1 and
SLC34A2 expression at the jejunal and ileal mucosa, respectively. This observation was supported by the increase in ΔI
SC as a response of the jejunal tissue to the added Na
2HPO
4 for pigs fed the LA-treated cereals in the Ussing chamber experiment, showing increased carrier-mediated cotransport of sodium ions and consequently of phosphate and thereby confirming greater jejunal P availability and absorption. Moreover, the LA-treated cereals seemed to increase the net absorption of anions, potentially phosphate, as indicated by the more negative inwardly directed I
SC [
81] in the Ussing chamber experiment. Albeit in a different intestinal segment, it can be speculated whether an increased paracellular Ca uptake mediated by higher caecal fermentation may have contributed to this enhancement in phosphate transporter expression. In line with that, the lower mucosal expression of the Ca channel
TRPV6 in the jejunum of pigs fed the diets containing the LA-treated cereals may be seen as a feedback control mechanism to reduce intestinal Ca uptake [
74,
82,
83]. The same reason may be true for the lower expression of
TRPV6 in the jejunum and ileum as well as of
TRPV5 (cecum and colon) and
TRPV6 (colon) in the large intestine of pigs receiving the phytase supplementation. Although the expression of the
VDR was not modified in a similar manner, adjustments in the VitD-regulated intestinal Ca uptake may have been further suggested by the lower mucosal expression of
CYP24A1 along the small intestine with phytase, potentially indicating less hydroxylation and thus degradation of active VitD.
To support our assumption with regards to the paracellular permeability and nutrient uptake, the mucosal-to-serosal flux rate of FITC-dextran indicated a largely increased jejunal permeability in pigs fed the LA diet, allowing more molecules to cross the mucosa. Fittingly, the serum Ca levels showed a similar profile as a response to the diets compared to the FITC-dextran flux rates (r = 0.44;
p = 0.016), supporting a potential link between paracellular Ca uptake and intestinal permeability. Since pigs were on the diet for 19 days, it is difficult to deduce whether these observations may have been either directly related to the LA diet, microbial action of the diet, or indirectly to systemic adaptations, which then may have led to the greater mucosal permeability. However, a greater paracellular jejunal uptake of Ca with the LA diet might be an explanation for the elevated serum Ca in pigs fed this diet. The gene expression results for tight-junction proteins indicated that tight-junction protein expression was influenced by the LA diet, more in the caecal and colonic mucosa, but inconsistently along the small and large intestinal segments. Except for the trend of a downregulated
CLDN4 expression in the jejunum, these data did not correspond to the measured FITC-dextran fluxes. This underlines that the results at the gene expression level do not necessarily explain the functional observations. Nevertheless, there was a general trend that the phytase supplementation downregulated the colonic expression of
ZO1 and
OCLN. These findings may correspond to the lower
TRPV5 and
TRPV6 expression in the colon with these diets, again hinting at a coupling of trans- and paracellular Ca transport [
16,
17,
84]. Notably, phytase supplementation reduced mucosal
MUC2 (caecum and colon) and
MUC4 expression (caecum). The question arises here whether this observation was due to changes in the microbial community caused by the varying Ca and P availability in these segments [
29,
85] or in relation to differences in the luminal Ca concentrations [
86,
87]. Accordingly, the elasticity of the mucus network formation depends on the intestinally available Ca; therefore, lower Ca availability in the large intestine might have led to negative feedback on
MUC2 and
MUC4 expression [
87].
For the serum parameters, it is vital to consider that pigs were not fasted when blood samples were collected but were in the resorptive phase two to three hours postprandial. Therefore, changes seen in serum parameters can be partly related to alterations in nutrient absorption and partly to long-term systemic adaptations to the diets. Overall, serum Ca and P levels emphasized the impact of the dietary Ca and P supply on the Ca and P metabolism as indicated by the Ca:P ratios in the serum, feces, and urine. If the dietary Ca supply is low, the body usually compensates this by the activation of VitD to increase intestinal absorption, renal resorption, and mobilization of Ca from the bones [
8,
43,
88]. Pigs consumed similar amounts of feed per meal, which implies similar VitD intake of all pigs. Therefore, changes in the serum levels may be compensatory actions to adjust the serum Ca levels. Accordingly, pigs fed the phytase-containing diets had higher serum VitD levels, which may have caused the lowered
OCN expression in the metacarpal bones, indicating reduced osteogenesis with the added phytase to balance serum Ca levels in the young growing animals. Likewise, increased renal expression levels of
VDR,
TRPV5, and
CALB1 would support an increased VitD-related resorption of Ca in pigs fed the phytase-containing diets. Calcitriol also stimulates FGF23 secretion from osteocytes [
89,
90]. However, the expression levels of
FGF23 in the marrow of the metacarpal bones and FGF23 levels in serum showed different dietary responses compared to serum VitD. There was a lowering effect of the LA-treated cereals on the
FGF23 expression in the bone, which corresponded to the lower expression of the VitD receptor and hence indicated decreased VitD signaling in the bone with the LA-treated cereals. However, there were no dietary treatment effects on the renal phosphate transporter expression to adjust renal P excretion [
89,
91], suggesting a different regulation.
FGF23 plays an essential part in P homeostasis, with the main regulating organs being bone, intestine, and kidney [
1,
2,
5]. Therefore, serum FGF23 may reflect regulatory action in relation to P homeostasis in the aforementioned organs. In the current study, serum FGF23 levels corresponded to the serum Ca levels, Ca/P ratio in serum, and urinary Ca excretion, being the highest in pigs fed the LA diet, and therefore seemed to be regulated by Ca rather than by P, which would also be supported by the present correlations. We would have assumed to find less FGF23 in serum of the pigs fed the LA diet in order to moderate the P excretion via the kidneys to enhance the utilization of Ca and P for osteogenesis. Instead, we found an increased urinary Ca excretion with the LA diet. So far, the relation between dietary Ca absorption and serum Ca and FGF23 levels is poorly understood [
90]. Before, serum FGF23 has been positively associated with dietary P availability [
92,
93], which was not the case in our study. Results from human hemodialysis patients, however, support a strong positive association between serum Ca and FGF23 [
94], which needs further investigation in order to complete our current understanding with respect to the underlying physiological signaling.
Due to the close correlation of bone-specific ALP and serum ALP, this biomarker is used in diagnosing osteomalacia in humans [
95,
96]. This disease is characterized by hypophosphatemia, which leads to a compensatory upregulation of ALP in order to increase free P [
2,
96]. Our findings demonstrated a positive correlation between serum levels of ALP and FGF23, suggesting that two opposing factors for the regulation of the P homeostasis were equally regulated. In fact, FGF23 has been reported to suppress ALP transcription [
97]. Interestingly, the metric bone data showed a different picture compared to the gene expression data, indicating a non-efficient use of Ca and P for the bone mass (i.e., thickness and area) but without affecting the actual mineral concentrations in bones, when dietary treatments were fed as single treatments but not when fed together. More efficient utilization of Ca and P with both dietary treatments would be also supported by the serum FGF23 level and the Ca and P retention, which were the lowest and highest when both treatments were combined, respectively. Despite the effect on the ash content due to both treatments, the CaO and P
2O
2 content of the bone ash were little affected by the dietary treatments, which shows that measuring the mineral concentrations in bones is not sufficient, even in young growing animals, to reliably predict diet-related alterations in mineral homeostasis and bone integrity. Therefore, the differences in ash content could have been caused by varying the fat and water associated with nutrition because the bones were not defatted for analysis [
98].
Taken together, as we assumed, both dietary treatments were efficient in improving the intestinal P availability and P retention in the present fast-growing pig model. Nevertheless, while phytase supplementation improved the retention of both Ca and P and the LA-treated cereals that of P, this came at the expense of reduced bone integrity, showing imbalances in mineral homeostasis, which was only counteracted when both treatments were combined. This may illustrate the sensitiveness of the mineral homeostasis towards imbalances in the dietary Ca and P uptake, particularly if this condition exists for a certain time (here three weeks). According to the present data and correlations, we may assume an imbalanced intestinal Ca and P absorption as the cause for differences in the activation of regulatory hormones and subsequently of the enzymatic machinery in the kidney and bones. Although VitD, FGF23, and ALP have been used as markers for mineral imbalances in the skeleton, only the weak correlation of FGF23 with serum Ca and the dietary Ca:P ratio may be helpful to link the systemic mineral availability with the dietary intake. Moreover, the results suggested that serum VitD levels are a useful indicator to predict the bone ash content and Ca resorption activity in the kidney, at least at the gene expression level, but this was irrespective from serum Ca or P. Lastly, due to the missing link between dietary P availability, serum FGF23, and P homeostasis, the present results also implicate the need for further research in relation to bone integrity markers for growing individuals. However, although growing pigs resemble adolescent humans in many physiological traits, it should be mentioned here that certain species-specific differences may exist, demanding for a verification of the observed relationships between dietary and serum parameters in humans.