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Oxford University Press

10.1093/jbmrpl/ziae075
ziae075
Research Article
AcademicSubjects/MED00010
AcademicSubjects/MED00160
AcademicSubjects/MED00250
Inorganic phosphate additives in meals and adaptations to 5-days of dietary inorganic phosphate loading alter acute calcium homeostasis in two randomized cross-over studies in healthy adults
https://orcid.org/0000-0001-6431-5077
Turner Mandy E Department of Biomedical and Molecular Science, Queen’s University, Kingston, ON K7L 2V7, Canada

Mazzetti Tom Department of Medicine, Queen’s University, Kingston, ON K7L 2V6, Canada

Neville Kathryn Department of Biomedical and Molecular Science, Queen’s University, Kingston, ON K7L 2V7, Canada

Ward Emilie C Department of Biomedical and Molecular Science, Queen’s University, Kingston, ON K7L 2V7, Canada

Munroe Jenny Department of Dietetics, Kingston Health Sciences Center, Kingston, ON K7L 2V7, Canada

Adams Michael A Department of Biomedical and Molecular Science, Queen’s University, Kingston, ON K7L 2V7, Canada

Holden Rachel M Department of Biomedical and Molecular Science, Queen’s University, Kingston, ON K7L 2V7, Canada
Department of Medicine, Queen’s University, Kingston, ON K7L 2V6, Canada

Corresponding author: Rachel M. Holden, Department of Medicine, Queen’s University, 3048C Etherington Hall, Kingston, ON K7L 2V6, Canada (rachel.holden@kingstonhsc.ca).
8 2024
06 6 2024
06 6 2024
8 8 ziae07505 4 2024
01 5 2024
06 7 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

Diets containing inorganic phosphate additives are unbalanced with respect to calcium and these diets have been linked to the development of altered bone metabolism. Using 2 randomized cross-over studies in healthy humans, we (1) characterized the hormonal and urinary response to 2 meals with the same reported phosphorus amount (562–572 mg), where one was manufactured with inorganic phosphate additives and a comparatively lower Ca:P molar ratio (0.26 vs 0.48), and (2) assessed how acute homeostatic mechanisms adapt following 5-d exposure to recommended dietary phosphorus amount (~700 mg P/d) compared to a diet enriched with inorganic phosphate additives (~1100 mg P/d). Participants were then challenged with 500 mg of oral phosphorus in the form of inorganic phosphate after an overnight fast following each diet condition. Measurements included serum calcium, phosphate, PTH, and fibroblast growth factor 23 , vitamin D metabolites, and urine calcium and phosphate excretion. Following the meal containing inorganic phosphate additives with a low Ca:P ratio, serum phosphate was higher and more phosphate was excreted in the urine compared to the low additive meal. Although the Ca:P and calcium content was lower in the high additive meal, the same amount of calcium was excreted into the urine. Subsequently, increasing only dietary phosphate through additives resulted in lower 24-h excretion of calcium. The oral phosphate challenge promoted urinary calcium excretion, despite no consumption of calcium, which was attenuated when pre-acclimated to a high phosphate diet. These data suggest that ingestion of inorganic phosphate promotes calcium excretion, but homeostatic mechanisms may exist to reduce calcium excretion that are responsive to dietary intake of phosphate. Future studies are required to evaluate potential implication of diets enriched with inorganic phosphate additives on bone health.

Graphical Abstract

Graphical Abstract

dietary phosphorus
phosphate
calcium
PTH
fibroblast growth factor 23
OPKO Health Inc. Renal Division Canadian Institutes of Health Research Vanier Scholarship
==== Body
pmcIntroduction

In healthy individuals, serum phosphate levels remain within the normal range despite wide fluctuations in dietary consumption due to homeostatic countermeasures that increase phosphate excretion into the urine. In healthy adults, the recommended dietary allowance (RDA) for phosphate is 700 mg of phosphorus/d; yet data from the National Health and Nutrition Examination Survey indicate that the average phosphorus intake in adults is nearly twice that, at 1368 mg of phosphorus/d.1 The terms “phosphate” and “phosphorus” are often used interchangeably; however in discussions of food amount in milligrams, it is important to make the distinction that phosphorus (P), the element, which is typically used in nutrition guidelines, and phosphate (PO4), the exclusive phosphorus moiety found in organic compounds, have different molecular weights and therefore are not directly comparable in milligrams.

Phosphate homeostasis is regulated via a complex interplay of intestinal absorption, urinary excretion, and movement of phosphate to and from extra-circulatory spaces, such as bone and intra/extracellular spaces in tissues.2,3 Acute homeostatic responses following consumption and longer-term compensatory mechanisms function together to maintain balance.2 Transient post-prandial elevations in serum phosphate cause PTH secretion from the parathyroid gland.4 PTH induces the conversion of 25OHD to 1,25(OH)2D3, which, along with phosphate directly, stimulates fibroblast growth factor 23 (FGF-23) production from osteocytes and osteoblasts.5 Sustained elevations in PTH increase bone resorption, resulting in the release of calcium and phosphate into the circulation, which is compensated for by the phosphaturic actions of PTH and FGF-23.6 Studies in humans have shown that the urinary excretion of an acute phosphate load, whether given orally or intravenously, is dependent on an acute PTH response.7,8

Phosphorus content is not required to be reported on nutrition labels, and nutrient databases typically underestimate phosphorus amounts in food, likely due to inaccurate/unavailable knowledge of additives.9 Up to 50% of total phosphorus intake has been estimated to be derived from inorganic phosphate additives.10 Further challenges to fully understanding phosphate consumption are stark differences in source-dependent bioavailability. Organic phosphate derived from food varies substantially in its bioavailability (~40%–60%); however, inorganic phosphate additives are highly bioavailable (~90%).11 Further, diets containing inorganic phosphate additives typically have low ratios with calcium (Ca:P), which impact on hormonal responses due to the co-regulation of these 2 minerals and co-storage as hydroxyapatite.12

Rigorous studies evaluating the relationship between long-term dietary phosphate intake, specifically with inorganic additives, and important clinical outcomes, such as osteoporosis and other mineral bone disorders or cardiovascular disease (CVD), in humans are lacking.13 Current evidence suggests that the use of dietary phosphate additives may have a negative impact on bone health. Pre-clinical studies have shown that diets with a low Ca:P ratio reduce bone mass and that PTH has a key role, as this effect can be prevented by prior parathyroidectomy.10,14,15 Further, in experimental animal studies, inorganic phosphate additives have a detrimental effect on bone and mineral metabolism despite not altering serum phosphate levels.10 Short intervention studies in humans confirm that a high phosphate (HP) diet often chronically increases PTH (4 d–4-wk intervention), especially when the Ca:P ratio of the diet is low,13,16-18 and increases FGF-23.17,18

Thus, it is prudent to carefully understand the acute (ie, post-prandial) and longer term homeostatic mechanisms at play in these scenarios of highly bioavailable phosphates consumed without proportionate calcium. Although several historical studies have investigated the role of dietary phosphate more generally, far fewer studies have focused on acute meal responses and impacts of chronic phosphate loading on acute responsiveness to phosphate challenges, especially in the last 30 years as our understanding of phosphate metabolism has changed substantially.13 Previous studies by our group have shown that assessment of circulating and urinary responses to a challenge can reveal differences in mineral homeostasis that is not identifiable with single time-point measurements.19,20 Using 2 randomized cross-over studies in young healthy adults, the objectives of the current study were: (1) to characterize the hormonal and urinary response to 2 meals with the same reported phosphorus amount but one manufactured with inorganic phosphate additives and a lower Ca:P ratio and (2) using an oral phosphate challenge, assess how acute homeostatic mechanisms adapt following 5 d of exposure to inorganic phosphate additives compared to an RDA diet.

Materials and methods

Study 1: Comparison of low and high phosphate additive meals

Healthy young adult female and male volunteers above the age of 18 were recruited from the community in Kingston ON, Canada (N = 18). All participants gave informed consent according to the Declaration of Helsinki and the study protocols were approved by Queen’s University and Associated Teaching Hospitals Research Ethics Boards. In a randomized cross-over design, participants consumed 2 breakfast meals after an overnight fast, and circulating minerals, hormones, and mineral urinary excretion were assessed for 3 h (Figure 1A). Participants were excluded if they were unable or unwilling to provide consent, were pregnant or breastfeeding, had a known allergy to any of the foods used in the breakfast meals, or had a history of impaired bladder emptying or kidney disease. All studies were conducted in the summer.

Figure 1 Study design of 2 randomized cross-over studies in young healthy adults. Study 1: phosphate additive meal comparison (A). The acute circulating mineral and urinary response was evaluated following 2 meals with the same reported phosphorus content, but one manufactured with inorganic phosphate additives. Study 2: dietary phosphate intervention. (B) Following 5-d of a dietician designed diet, participants completed an OPTT protocol. One diet contained the RDA of phosphate, and the HP contained the typical Western dietary intake. Following 5 d of each diet, fasted participants consumed a drink containing 500 mg phosphorus. Blood and urinary responses were assessed hourly for 3 h. A subset of 5 participants completed the protocol without the OPTT presented in supplementary figures. The randomized cross-over design of both studies included an interim period of at least 7 d.

Two meals were prepared which contained the same amount of reported phosphorus. One meal was composed of foods containing phosphorus with lower bioavailability, and the other “convenience” meal was composed of processed foods that contained inorganic phosphate additives (higher bioavailability). The compositions of the meals are shown in Table S1. Nutritional values were obtained from the product’s nutritional facts reported by the provider. Values for phosphorus and calcium composition of the “convenience meal” were confirmed using in-house colorimetric assays. Several samples from each food item were placed in 1 N HCl per weight, homogenized, shaken at room temperature for 24 h, centrifuged (12 000 g for 20 min), and the aqueous top layer was measured. Calcium was measured using the o-cresolphthalein complexone method at 540 nm (Sigma-Aldrich, CAN) as previously described.21 Free phosphate was measured using the malachite green (Sigma-Aldrich, CAN).22 Both test meals consisted of 562–572 mg P.

The study used a randomized cross-over design on 2 different mornings separated by at least 7 d. Participants performed a 24-h urine collection starting the morning before testing. The participants began the experiment at 07:30 after fasting from 19:30 the previous night. The 2 test meals were served in random order. The food consumption was completed within 15 min, and subjects could drink water freely thereafter. Serum and plasma measurements were performed for each test meal pre-prandial (0 minutes) and postprandial (30, 45, 60, 75, 90, 120, and 180 min). Urine measurements were performed for each test meal pre-prandial and postprandial (60, 120, and 180 min).

Statistical analysis for study 1

Boxplots indicate median, IQR, and min/max. Data are represented as raw values. Fractional excretion at each time point was calculated as FEPhos = (UPhos × SCr)/(UCre × SPhos) or FECa = (UCa × SCr)/(UCre × SCa). Timed differences in response to meals were evaluated using a matched mixed-effects model with post hoc Sidak-corrected paired T-tests evaluating differences within each diet from baseline and between diets at each timepoint. A 2-way ANOVA was not possible due to data missing at random. All statistical tests and graph generation were done on IBM SPSS Statistics (Version 20) or Graph Pad Prism (Version 10).

Study 2: Acute homeostatic phosphate response following 5-d dietary intervention of RDA or dietary high in phosphate additives

Healthy young adult female and male volunteers above the age of 18 were recruited from the community in Kingston, ON, Canada (N = 14). All participants gave informed consent according to the Declaration of Helsinki and the study protocols were approved by Queen’s University and Associated Teaching Hospitals Research Ethics Boards.

In a randomized, cross-over design, participants were provided with a 5-d meal plan to be followed during both arms of the trial that reflected the RDA for phosphorus intake (701–742 mg phosphorus/d, RDA). During the HP arm, an additional 400 mg phosphorus supplement in the form of inorganic phosphate taken in 2 divided doses in the morning and evening was provided for a total daily phosphorus intake of 1100–1142 mg/d (Table S2). The study design is presented in Figure 1B. Thus, each participant completed 2 trials, with at least a week between trials, during which time participants returned to their normal diet.

Participants were requested to complete a comprehensive food diary during both test periods. The prescribed food plan was developed by a registered dietitian. Meals were not provided. This intake record was cross-referenced with the Canadian Nutrient File by a registered dietitian to compare estimated phosphorus intake with prescribed. The recommended meal plan contained 269–321 mg of dietary calcium/d. A calcium supplement containing 500 mg elemental calcium was taken daily during both arms of the study, and thus the dietary calcium during each diet condition totaled 769–821 mg/d (RDA 1000 mg/d) as the diet during the HP arm was identical to the RDA arm. The calcium supplement was not taken at the same time as the 400 mg phosphorus supplement in the HP arm. Therefore, the prescribed dietary Ca:P molar ratio was approximately 0.85 in the RDA diet and 0.55 in the HP diet. The values are outlined in Table S2.

Following the dietary intervention, participants presented after an overnight fast and 24-h urine collection. Pre-OPTT baseline blood and urine samples were taken. Participants then completed an oral phosphate tolerance test (OPTT): they consumed a 500 mg oral phosphorus load (sodium-phosphate) within 5 min, and blood and subsequent urine samples were obtained hourly for 3 h. Participants remained fasted during the study.

After the randomized-cross over study was completed, a subset of 5 participants completed the protocol without the oral phosphate load. Following an overnight fast, serial measurements of blood and urine were taken at the same time points described above. Participants remained fasted during the study.

Statistical analysis for study 2

Boxplots indicate median, IQR, and max/min. Paired T-tests were performed to compare pre-oral challenge laboratory measures and area under the curve (AUC) values between the high and RDA dietary phosphate conditions or meals. Data are represented as raw values, absolute change from baseline (value–baseline value) where baseline is prior to the OPTT. Fractional excretion at each time point was calculated as FEPhos = (UPhos × SCr)/(UCre × SPhos) or FECa = (UCa × SCre)/(UCre × SCa). To evaluate changes in timed measurements, repeated measures matched 2-way ANOVA with post hoc Sidak-corrected paired T-tests evaluating differences within each diet from baseline and between diets at each timepoint. AUC was calculated using trapezoidal approximation. All statistical tests and graph generation were done on IBM SPSS Statistics (Version 20) or Graph Pad Prism (Version 10).

Correlations between FGF-23 and PTH with measures were assessed using Spearman r values. In tables, the values under each phosphate diet indicate the correlation between the values only measured after the indicated diet (ie, each participant’s FGF-23 value correlated with their 24-urine phosphate measured after the week of lower dietary phosphate). The difference value indicates the value on the HP diet minus the value on the RDA diet (ie, the extent to which FGF-23 changed in response to the HP diet correlated with the extent to which the 24-h urine phosphate changed). Delta AUC indicates net AUC of the absolute change graph. Negative change corresponds to a negative AUC.

Laboratory methods

In study 1, serum and urine creatinine, phosphate, and calcium were measured by the Roche Plus Modular assay, and PTH was measured by immunoassay on a Roche Modular E170 System (Roche Diagnostics, Indianapolis, IN) with an assay imprecision of <3%. These analytes were assessed in the core lab of Kingston Health Sciences Center. In study 2, serum and urine creatinine, phosphate, and calcium as well as PTH were measured as outlined above. Estimated glomerular filtration rate was calculated by the CKD-EPI equation.23 Serum vitamin D metabolites were measured by LC–MS/MS methods (Model Acquity LC/Xevo, TQ-S LC–MS/MS system; Waters Cor) using previously described methods.24,25 Plasma intact FGF-23 (Immunotopics) and Osteocalcin (Invitrogen) were measured in duplicate using ELISA as per the manufacturer’s instructions, with coefficients of variation <10% and R2 > 0.995.

Results

Study 1: Comparison of low and high phosphate additive meals

Eighteen participants, with mean age of 22 ± 2, completed both arms of the randomized cross-over meal study (11 females and 7 males) (Figure 1A). All participants were white. Following an overnight fast, 1 of 2 meals was consumed in random sequence. After the high inorganic phosphate additive meal with low Ca:P ratio, serum phosphate was higher than the low-additive meal between 45 min and 3 h (Figure 2A), and there was greater excretion of phosphate into the urine at 120 and 180 min (Figure 2B and C). Although both meals had the same amount of reported phosphorus, the HP additive meal had less calcium (190 mg, Ca:P ratio of 0.26) than the low additive meal (345 mg: Ca:P ratio of 0.48) (Table S1). Accordingly, serum calcium was lower following the HP additive meal 60, 120, and 180 min after the challenge; however, the urinary calcium excretion was not different (Figure 2D–F). PTH changed similarly following the meals (Figure S1). Given these differences in calcium response to a single meal, we sought to characterize physiological adaptations to dietary phosphate additive loading with a reduced Ca:P ratio.

Figure 2 HP additive meal results in higher serum phosphate and urinary excretion compared to a low additive meal with the same total amount of reported phosphorus (study 1). Timed changes in serum phosphate (A), urinary phosphate to creatinine ratio (UPhos:UCre) (B), fractional excretion of phosphate (C), serum calcium (D), urinary calcium to creatinine ratio (UCa:UCre) (E), and fractional excretion of calcium (F). Matched mixed effects model with post hoc paired T-tests evaluating differences within each diet from baseline (Sidak corrected, *p<.05, **p<.01, ***p<.001) and between diets at each timepoint (Sidak corrected, †p<.05,, †††p<.001). Data expressed as median, IQR with error bars indicating min/max.

Study 2: Assessment of acute homeostatic phosphate response using OPTT following 5-d dietary intervention of RDA or a diet high in inorganic phosphate additives

Participant characteristics and dietary changes

A total of 14 healthy participants completed the study protocol (Figure 1B). There were 9 females, 5 males, and the mean age was 23.3 ± 2.5. All participants were white. Both interventions were prescribed the same diet; however, during the HP additive diet period, participants consumed an additional inorganic phosphate drink twice daily, which was reflected in the marked increase in 24-h urine phosphate (Table 1). Using food diaries, the intake of phosphorus derived from food was similar during both diet protocols (811 ± 316 mg phosphorus/d for the RDA diet vs 814 ± 266 mg phosphorus/d for the HP diet), indicating adherence to the dietitian-recommended diet. Total dietary calcium intake was similar between the 2 diets as well: 450 ± 241 mg/d during the RDA diet and 471 ± 259 mg/d during the HP diet.

Table 1 Fasted urinary and circulating measures of human study participants following the RDA phosphate diet and the high inorganic phosphate supplemented diet prior to the OPTT.

	RDA phosphate diet	High phosphate diet	p-value	
Urinary measures		
24-h urine phosphorus (mg)	687.5 ± 353.1	1551.6 ± 473.8	<0.0001	
UPhos:UCre (mM/μM)	1.65 ± 0.75	2.94 ± 1.06	0.0005	
FEPhos (%)	9.4 ± 4.8	16.7 ± 4.4	0.0003	
24-h urine Ca (mg)	128.3 ± 76.1	92.2 ± 60.1	0.008	
UCa:UCre (mM/μM)	0.11 ± 0.07	0.12 ± 0.06	0.58	
FECa (%)	0.38 ± 0.22	0.38 ± 0.19	0.97	
24-h urine urea (mmol)	284.2 ± 170.7	292.2 ± 153.0	0.74	
24-h urine creatinine (μmol)	12.3 ± 5.2	12.4 ± 4.0	0.93	
24-h urine volume (mL)	1632 ± 825	1469 ± 858	0.70	
Blood measures		
Phosphate (mM)	1.21 ± 0.21	1.26 ± 0.16	0.43	
Calcium (mM)	2.36 ± 0.07	2.35 ± 0.07	0.53	
Magnesium (mM)	0.82 ± 0.03	0.80 ± 0.06	0.08	
Creatinine (μM)	77.8 ± 12.8	76.4 ± 11.9	0.48	
eGFR (mL/min/1.73 m2)	104.2 ± 15.6	106.3 ± 13.3	0.49	
PTH (pmol/L)	3.80 ± 1.23	4.59 ± 1.45	0.03	
FGF-23 (pg/mL)	69.2 ± 48.2	81.9 ± 45.4	0.002	
Osteocalcin (ng/mL)	5.94 ± 2.82	6.6 ± 2.58	0.24	
25(OH)D3 (ng/mL)	29.8 ± 9.1	28.7 ± 9.2	0.55	
24,25(OH)2D3 (ng/mL)	1.9 ± 0.8	2.1 ± 0.9	0.29	
24,25(OH)2D3:25(OH)D3	0.063 ± 0.015	0.071 ± 0.012	0.03	
1,25(OH)2D3 (pg/mL)	42.6 ± 12.8	38.8 ± 11.0	0.33	
Data presented as mean ± SD. Paired T-test between dietary phosphate conditions. Without participant with high FGF-23, circulating FGF-23 averages are 57.3 ± 14.7 vs 71.3 ± 20.1** for RDA and HP diets, respectively.

Bolded values indicate p <0.05.

Abbreviations: eGFR, estimated glomerular filtration rate; FGF-23, fibroblast growth factor 23; RDA, recommended dietary allowance.

Biochemical changes induced by diet

Following the 5-d dietary intervention, participants arrived fasted for pre-OPTT baseline measurements. Baseline UPhos: UCre and FEPhos were significantly higher after the HP diet compared to the RDA diet (Table 1). The 24-h urine calcium was significantly lower following the HP diet, but pre-OPTT baseline UCa:UCre and FECa were similar. PTH and FGF-23 significantly increased after the HP diet, but baseline serum levels of phosphate and calcium were unchanged. Osteocalcin, a marker of bone formation, was not altered by the diet. One participant had abnormally elevated levels of FGF-23 at both time points (230–235 pg/mL compared to 39–118 pg/mL in the other participants), with no other discordant circulating or urinary parameters. This individual had a decrease in FGF-23 in response to the HP diet. All correlative assessments were completed with and without this participant, indicated in Table S3. There was no difference in 25(OH)D3 or 24,25(OH)2D3 levels between diets; however, the 24-hydroxylation ratio between 24,25(OH)2D3 and 25(OH)D3 increased following the HP diet, suggesting enhanced 25(OH)D3 catabolism.

OPTT: Post-challenge phosphate handling

There was no difference in serum phosphate between diet conditions at any time point following the OPTT challenge (Figure 3A). The change in UPhos:UCre following the 500 mg challenge was the same after each diet, indicating similar capacity for acute excretion of phosphate (Figure 3B). FEphos increased by a similar amount following the HP and the RDA diets in response to the oral phosphate load; however, the relative increase in FEPhos was higher following the RDA diet (Figure 3C). In participants who did not consume the inorganic phosphate challenge after an overnight fast, serum phosphate, UPhos:UCre, and FEPhos did not change from baseline (Figure S2A–F).

Figure 3 High dietary phosphate does not alter the acute profile of urinary excretion of phosphate following an oral phosphate load in humans (study 2). (A) Timed values of serum phosphate, absolute change of serum phosphate from baseline, and relative change of serum phosphate from baseline, (B) urinary phosphate to creatinine ratio (UPhos:UCre), absolute change of UPhos:UCre from baseline, and relative change of UPhos:UCre from baseline, (C) fractional excretion of phosphate (FEPhos), absolute change of FEPhos from baseline, and relative change of FEPhos from baseline. Repeated measures 2-way ANOVA with post hoc paired T-tests evaluating differences within each diet from baseline (Sidak corrected, *p<.05, **p<.01, ***p<.001, ***p<.0001) and between diets at each timepoint (Sidak corrected, ††p<.01, †††p<.001). Data expressed as median, IQR with error bars indicating min/max.

OPTT: Post-challenge calcium handling

There was no difference in serum calcium between diet conditions at any time point following the challenge (Figure 4A). Calcium excretion increased post-challenge following both diet conditions but was greater following the RDA diet (Figure 4B). The area under the absolute change UCa:UCre curve was smaller by 39% following the HP diet (0.33 ± 0.22 mM/μM × hr vs 0.20 ± 0.22 mM/μM × hr, p=.02, Table S4). The absolute and relative change in FECa increased in response to the oral phosphate challenge (Figure 4C). The area under the absolute change FECa curve was smaller by 57% following the HP diet (1.1 ± 0.6 % × hr vs 0.7 ± 0.6% × hr, p=.04, Table S4). In the participants who did not consume the phosphate challenge after an overnight fast, UCa:UCre, and FECa did not change from baseline (Figure S3A–F).

Figure 4 High dietary phosphate attenuates the acute urinary excretion of calcium following an oral phosphate load in humans (study 2). Timed values of (A) serum calcium, absolute change of serum calcium from baseline, and relative change of serum calcium from baseline, (B) urinary calcium to creatinine ratio (UCa:UCre), absolute change of UCa:UCre from baseline, and relative change of UCa:UCre from baseline, (C) fractional excretion of calcium (FECa), absolute change of FECa from baseline, and relative change of FECa from baseline. Repeated measures 2-way ANOVA with post hoc paired T-tests evaluating differences within each diet from baseline (Sidak corrected, *p<.05, **p<.01, ***p<.001, ***) and between diets at each timepoint (Sidak corrected, †p<.05, p<.01, p<.001). Data expressed as median, IQR with error bars indicating min/max.

Post-challenge PTH

Serum PTH remained constant in subjects pre-acclimated to the RDA diet and decreased from approximately 4 pmol/L to 3 pmol/L in subjects pre-acclimated to the HP diet (Figure 5). The cumulative difference in serum PTH (from pre-challenge baseline), as indicated by the area under the change in PTH curve, was significantly greater after the RDA diet compared to the HP diet despite being negative (−0.40 ± 2.31 pmol/L × hr vs −2.26 ± 2.81 pmol/L × hr, p<.05, Table S4). In the participants who did not consume the phosphate challenge after an overnight fast, PTH did not significantly change from their fasted baseline value (p=.06), although trended downwards to a greater extent than either diet group (Figure S4A–C).

Figure 5 High dietary phosphate alters the acute profile of PTH following an oral phosphate load in humans (study 2). Timed values of serum PTH, absolute change of serum PTH from baseline, and relative change of serum PTH from baseline. Repeated measures 2-way ANOVA with post hoc paired T-tests evaluating differences within each diet from baseline (Sidak corrected, *p<.05, p<.01, ***p<.001) and between diets at each timepoint (Sidak corrected, †p<.05, ††p<.01, p<.001). Data expressed as box plot with error bars indicating min/max.

Correlations of phosphate and calcium handling with PTH and FGF-23

FGF-23 and PTH were strongly associated with each other following the HP diet only (r = 0.71, p<.01, Table S5). The AUC for absolute change in serum phosphate was calculated as an overall measure of the impact of newly introduced phosphate on serum phosphate. The more that FGF-23 increased in response to the HP diet, the less serum phosphate acutely increased (r = −0.50, p=.07). However, FGF-23, or the change in FGF-23 between diets, was not associated with markers of urinary excretion: the AUC for the absolute change in Uphos:Ucre or FEphos.

Following the RDA diet, fasted pre-OPTT baseline PTH was positively associated with the AUC for the absolute change in phosphate excretion (r = 0.7, p<.05). The association between PTH and urinary phosphate excretion disappeared following the HP diet condition where baseline levels of PTH were higher. The more that PTH increased in response to the HP diet, the less that PTH levels changed acutely in response to the phosphate challenge, as indicated by a negative correlation between PTH and the AUC of the serum PTH change curve (r = −0.74, p<.01).

The change in serum phosphate at 1 h and the AUC of the change in serum phosphate correlated with the absolute change in PTH at 1 h following both diets (r = 0.53–0.87) (Table S6). Acute changes in calcium did not correlate in either dietary setting to the PTH response.

Discussion

Using 2 randomized cross-over studies, we assessed (1) the acute response to 2 meals with differing inorganic phosphate additives but the same reported total phosphorus content and (2) the adaptation of acute phosphate homeostasis to 5 d of a diet enriched with inorganic phosphate additives in healthy young adults. The major finding of this study was the impact of both the presence of additives in a meal and 5-d loading on acute calcium homeostasis. As the intake of convenience foods containing phosphate additives increases, research will need to determine whether these diets influence bone health in a way that may not become apparent until later in life.

In the first study (study 1), we confirmed that a meal containing bioavailable phosphate additives resulted in higher serum and urine excretion of phosphate, compared to a meal containing the identical reported phosphorus but of lower assumed bioavailability. Consistent with known food trends, the high additive meal contained less calcium and, as a result, a lower Ca:P ratio.12 Although the high additive meal had lower calcium content, the same amount of calcium was excreted into the urine over the next 3 h and serum calcium decreased in response to this meal. These data suggest that, at least over the course of 3-h experimental period, calcium was preferentially retained in the setting of a meal with a higher Ca:P ratio, or alternatively, excretion into the urine of calcium from an endogenous source occurs following meals enriched with phosphate additives. Both could impact overall calcium balance and bone stores; however, we also cannot rule out the possibility that the meal composition differences impacted calcium absorption from the small intestines. Compartmental modelling of calcium and phosphate homeostasis suggests the bone has both a rapidly exchangeable pool of calcium and phosphate as well as a more permanent slowly exchangeable pool.2 It was therefore relevant to assess whether compensatory mechanisms modulating acute responses adjust for longer term consumption of diets high in phosphate additives with a lower Ca:P ratio in healthy individuals.

A 5-d dietary intervention was completed that compared an RDA diet to a high inorganic phosphate diet with the same amount of calcium that was chosen to reflect the dietary composition prevalent in North America. The 24-h excretion of calcium was significantly reduced following the HP diet. A meta-analysis investigating alkaline diets reported that inorganic phosphate supplementation was associated with decreased urine calcium, consistent with our findings.26 Furthermore, calcium isotope balance studies of increased dietary phosphate loading with fixed calcium also report reduced urinary calcium excretion and rule out reduced intestinal calcium absorption as the cause at calcium intakes below 2000 mg/d.27 However, Gutierrez et al. and Antonucci et al. reported non-reduced 24-h urine calcium excretion following 7 and 9 d of an additive-supplemented diet, respectively.10,18 PTH has a recognized role in promoting renal calcium reabsorption, and there is increasing evidence to suggest that FGF-23, in addition to being a phosphotonin, also promotes renal calcium reabsorption through the TRPV5 channel.28 Vitamin D is a calcium and phosphate regulating hormone with its principal role being the promotion of intestinal calcium absorption.29 Serum levels of both 25(OH)D3 and 1,25(OH)D3 were not altered in response to the dietary phosphate intervention; however, the ratio of 25(OH)D3 to its catabolic metabolite, 24,25(OH)2D3, (24,25(OH)2D3:25(OH)D3), was increased, indicative of a potentially more catabolic state of vitamin D metabolism. Consistent with this finding, the enzyme which regulates this conversion, CYP24A1, is upregulated by FGF-23.30 These findings suggest that the observed reduction in 24-h urine calcium in response to dietary phosphate is unlikely to be a result of hormonally regulated calcium absorption in the intestine. The decrease in the 24-h excretion of calcium that we observed following the HP diet in study 2 may represent an adaptive response of the body to retain calcium in the setting of a diet where phosphate is high and unbalanced with respect to calcium.

In the dietary intervention study (study 2), the acute homeostatic response to phosphate was assessed using an OPTT where participants consumed a drink containing only inorganic phosphate. This challenge triggered a response resulting in calcium excretion, suggesting either a direct or indirect effect of inorganic phosphate on calcium release from bone or soft tissues. Calcium excretion was attenuated when participants were pre-acclimated to an HP diet, indicating that adaptive mechanisms to chronic dietary phosphate additives exist that retain calcium. The response of serum calcium and phosphate to the challenge was identical between diets; however, the PTH response from their basal fasted values differed. Although the level of PTH was sustained in response to the challenge following the RDA diet, it decreased following the HP diet. It is possible that an increase in PTH may have been seen if we had observed a larger elevation of serum phosphate in response to the challenge, as has been previously reported.19 Our finding of stable or decreasing PTH must be interpreted in light of the known PTH diurnal rhythm that exists in humans where the nadir of daily PTH is observed around mid-day, with reductions between 8 AM and noon being consistently reported, as was reflected in our controlled participant group that did not consume the phosphate challenge and has been observed in other acute phosphate loading studies.31-34 Further, dietary phosphate has been shown to not impact the circadian rhythm of PTH in healthy humans,31,33 and is therefore not a likely explanation for the different PTH response. One potential hypothesis is that resistance to CaSR-mediated signaling develops in the PTG in response to dietary phosphate. A downregulation of CaSR in the PTG in response to a HP diet has been shown in some35 but not all studies.36 In our study, the 1-h change in circulating phosphate was strongly correlated with the 1-h change in PTH regardless of the dietary pre-acclimation. Overall, the lower the pre-OPTT fasted baseline phosphate, the more responsive PTH was to the phosphate challenge, whereas calcium changes were not similarly correlated. An alternative hypothesis is that an efficient homeostatic response exists that is acutely independent of PTH.

Taken together, these studies identify a role of inorganic phosphate in altering calcium homeostasis, acutely after a single meal and more chronically following 5-d adaptation to diets, which could have implications for bone health and requires further exploration. Gutierrez et al. demonstrated that consumption of 2 diets, similar in calcium but increased inorganic phosphate, increased bone biomarkers of resorption (osteopontin) and formation (osteocalcin), and reduced levels of P1NP, a marker of formation, in healthy humans after only 1 wk.10 Parallel studies conducted in mice demonstrated unfavorable changes in cortical and trabecular bone geometry after 5 wk of high dietary phosphate.10

The capacity to excrete phosphate acutely into the urine following a phosphate load was not altered by the 5-d dietary intervention. The higher fasted pre-OPTT baseline levels of PTH and FGF-23 following the HP diet, which act to decrease kidney tubular reabsorption of phosphate through NaPi2a/2c, are consistent with the higher baseline FEphos. These data suggest that, in healthy adults with normal kidney function, the renal response to an acute phosphate load is maintained and not altered by previous dietary phosphate exposure, or at least not at this level of intake over 5 d and acute exposure.

The potential for differences in gastrointestinal absorption of the challenge post HP diet to explain some findings should be considered. Ex vivo studies assessing intestinal phosphate absorption need to be considered carefully in light of increasing numbers of manuscripts using in situ or in vivo methods published contradicting previous ex vivo findings.37 In a study, dietary level of phosphate did not affect either phosphate absorption in the jejunum in situ or Na-Pi 2b gene expression in healthy rats.38

The HP diet in our study mirrors the typical diet in North America, comprised of processed foods rich in highly bioavailable inorganic phosphate additives with relatively less calcium. The amount of phosphate contained in our oral challenge approximates the amount of phosphate contained in a processed food meal,39 as indicated by the meal study (study 1). The randomized cross-over study designs are a significant strength as they allow for rigorous assessment of changes in phosphate handling as a result of the interventions, accounting for wide inter-participant heterogeneity due to various characteristics, known and unknown, that may influence phosphate homeostasis, such as estrogen and testosterone levels modulating kidney phosphate transporters,40,41 and other factors such as sex,20 ethnicity,42 and obesity.43 Providing a supplement to the standardized diet was chosen to facilitate rigorous adherence and nutritional similarity between diets. We examined young healthy individuals which limits applicability to older people or those with impaired kidney function. The lack of diversity in ethnic backgrounds is a weakness. Ethnic differences in acute phosphate handling or post-prandial response have been previously reported,42,44 and thus our findings may not translate to other demographics and should be a focus of future research. Further, the study was conducted fasted at the beginning of the day, which is also the nadir for serum phosphorus. Although this facilitated similar basal levels and a baseline value unbiased by prior consumption, studies have shown not only a clear circadian rhythm and further that alterations in serum phosphate due to dietary consumption may be more clearly demonstrated later in the day. Lastly, the study was not powered to detect sex differences in response to dietary phosphate; however, we have previously reported sex differences in response to an OPTT in older adults.20

Adaptations in response to dietary phosphate intake maintain serum phosphate levels within the normal range and do not appear to alter the capacity to excrete phosphate acutely at this level of phosphate exposure. Calcium excretion occurs in response to a phosphate load or phosphate-containing meal; however, an adaptive shift to decrease urinary calcium excretion occurs once acclimated to a HP diet. These findings indicate an important role of inorganic phosphate additives on acute calcium homeostasis that will need to be carefully explored for potential implications on bone and/or vascular outcomes. Together these findings also indicate the importance of bioavailability of phosphate and ratio of phosphate to calcium in dietary management.

Author contributions

Mandy E. Turner (Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Visualization, Writing—original draft, Writing—review & editing), Tom Mazzetti (Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Writing—review & editing), Kathryn Neville (Data curation, Methodology, Writing—review & editing), Emilie C. Ward (Data curation, Formal analysis, Writing—review & editing), Jenny Munroe (Methodology, Writing—review & editing), Michael A. Adams (Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing—review & editing), and Rachel M. Holden (Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing—original draft, Writing—review & editing)

Funding

M.A.A. and R.M.H. were funded by OPKO Health Inc. Renal Division. unrelated to the current studies. M.E.T. was supported by Canadian Institutes of Health Research Vanier Scholarship.

Conflicts of interest

Study Design: M.E.T., T.M., K.N., J.M., M.A.A., R.M.H. Study Conduct: M.E.T., T.M., E.C.W., K.N. Data Collection: M.E.T., T.M., E.C.W., K.N. Data Analysis: M.E.T., K.N., M.A.A., R.M.H. Data Interpretation: M.E.T., M.A.A., R.M.H. Drafting Manuscript: M.E.T., R.M.H. Revising Manuscript and Content: M.E.T., M.A.A., R.M.H. Approving final version of manuscript: M.E.T., T.M., K.N., E.C.W., J.M., M.A.A., R.M.H. R.M.H. takes responsibility for the integrity of the data analysis.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary Material

Phosphate_diet_supplementary_20240530_ziae075
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