Acute effect of oral phosphate loading on serum fibroblast growth factor 23 levels in healthy men
2006; Elsevier BV; Volume: 70; Issue: 12 Linguagem: Inglês
10.1038/sj.ki.5002000
ISSN1523-1755
AutoresYuka Nishida, Yutaka Taketani, Hisami Yamanaka‐Okumura, Fumiaki Imamura, Atsuki Taniguchi, Tadatoshi Sato, Emi Shuto, Kunitaka Nashiki, Hidekazu Arai, Hironori Yamamoto, Eiji Takeda,
Tópico(s)Genetic Syndromes and Imprinting
ResumoSerum fibroblast growth factor 23 (FGF23) is a novel phosphaturic factor and important for the regulation of inorganic phosphate (Pi) homeostasis. In this study, we examined an acute effect of oral Pi loading on serum FGF23 levels to clarify the role in rapid adjustment of serum Pi level. We performed a randomized, double-blind, crossover study in eight healthy male volunteers. The subjects were alternately served one of three test meals containing different Pi amounts (400 mg (P400), 800 mg (P800), and 1200 mg (P1200)) as lunch at noon. The postprandial changes in serum levels of Pi, Ca, 1,25-dihydroxyvitamin D, intact-parathyroid hormone (iPTH), intact-FGF23 (iFGF23), and urinary excretion of Pi and Ca until 8 h after Pi loading were estimated. Serum Pi levels and urinary Pi excretion significantly increased within 1 h after P400 and P800 intake. Serum iPTH levels at 1–2 and 4–6 h after P1200 intake was significantly higher than those of P400 intake. Serum iFGF23 levels slightly decreased up to 8 h after P400 intake and up to 6 h after P800 intake, but not changed in P1200 intake. Significant increase of iFGF23 was observed at 8 h after P1200 intake compared with both P400 and P800 intake. Additionally, negative association was detected between iFGF23 and serum Pi, whereas positive association was observed between iPTH and serum Pi during the short period. We conclude that oral Pi loading cannot rapidly increase serum FGF23 level. FGF23 may be not associated with rapid adaptation of Pi homeostasis. Serum fibroblast growth factor 23 (FGF23) is a novel phosphaturic factor and important for the regulation of inorganic phosphate (Pi) homeostasis. In this study, we examined an acute effect of oral Pi loading on serum FGF23 levels to clarify the role in rapid adjustment of serum Pi level. We performed a randomized, double-blind, crossover study in eight healthy male volunteers. The subjects were alternately served one of three test meals containing different Pi amounts (400 mg (P400), 800 mg (P800), and 1200 mg (P1200)) as lunch at noon. The postprandial changes in serum levels of Pi, Ca, 1,25-dihydroxyvitamin D, intact-parathyroid hormone (iPTH), intact-FGF23 (iFGF23), and urinary excretion of Pi and Ca until 8 h after Pi loading were estimated. Serum Pi levels and urinary Pi excretion significantly increased within 1 h after P400 and P800 intake. Serum iPTH levels at 1–2 and 4–6 h after P1200 intake was significantly higher than those of P400 intake. Serum iFGF23 levels slightly decreased up to 8 h after P400 intake and up to 6 h after P800 intake, but not changed in P1200 intake. Significant increase of iFGF23 was observed at 8 h after P1200 intake compared with both P400 and P800 intake. Additionally, negative association was detected between iFGF23 and serum Pi, whereas positive association was observed between iPTH and serum Pi during the short period. We conclude that oral Pi loading cannot rapidly increase serum FGF23 level. FGF23 may be not associated with rapid adaptation of Pi homeostasis. Inorganic phosphate (Pi) is an essential nutrient for many biological processes, including skeletal mineralization and energy metabolism. Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption and renal excretion of Pi. These processes are primarily regulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)2D).1.Berndt T.J. Knox F.G. Renal regulation of phosphate excretion.in: Seldin D.W. Diebish G. The Kidney, Physiology and Pathophysiology. 2nd edn. Raven press, New York1992: 2511-2532Google Scholar, 2.Murer H. Biber J. Renal tubular phosphate transport.in: Seldin D.W. Diebish G. The Kidney, Physiology and Pathophysiology. 2nd edn. Raven press, New York1992: 2481-2509Google Scholar, 3.Murer H. Hernando N. Forster I. Biber J. Proximal tubular phosphate reabsorption: molecular mechanisms.Physiol Rev. 2000; 80: 1373-1409Crossref PubMed Scopus (424) Google Scholar However, PTH and 1,25(OH)2D act predominantly to control Ca homeostasis, and Pi homeostasis often undergoes changes as a result. Therefore, the regulation of Pi homeostasis is not clearly understood despite its broad biological importance. Recently, emerging evidence suggests that there are other factors of bone origin that participate in maintaining Pi homeostasis, such as fibroblast growth factor 23 (FGF23),4.Shimada T. Mizutani S. Muto T. et al.Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia.Proc Natl Acad Sci USA. 2001; 98: 6500-6505Crossref PubMed Scopus (1120) Google Scholar matrix extracellular phosphoglycoprotein,5.Rowe P.S.N. de Zoysa P.A. Dong R. et al.MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia.Genomics. 2000; 67: 54-68Crossref PubMed Scopus (301) Google Scholar and frizzled-related protein-4.6.Berndt T. Craig T.A. Bowe A.E. et al.Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent.J Clin Invest. 2003; 112: 785-794Crossref PubMed Scopus (226) Google Scholar Among these factors, FGF23 has been shown to be associated with the hypophosphatemic diseases, including autosomal-dominant hypophosphatemic rickets, tumor-induced osteomalacia, and X-linked hypophosphatemic rickets.7.Takeda E. Yamamoto H. Nashiki K. et al.Inorganic phosphate homeostasis and the role of dietary phosphorus.J Cell Mol Med. 2004; 8: 191-200Crossref PubMed Scopus (95) Google Scholar, 8.Berndt T.J. Schiavi S. Kumar R. 'Phosphatonins' and the regulation of phosphorus homeostasis.Am J Physiol Renal Physiol. 2005; 289: F1170-F1182Crossref PubMed Scopus (169) Google Scholar, 9.Yu X. White K.E. FGF23 and disorders of phosphate homeostasis.Cytokine Growth Factor Rev. 2005; 16: 221-232Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Shimada et al.10.Shimada T. Muto T. Urakawa I. et al.Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo.Endocrinology. 2002; 143: 3179-3182Crossref PubMed Scopus (354) Google Scholar have reported that administration of the recombinant FGF23 protein reduced serum Pi without affecting serum calcium, and also increased renal Pi excretion in mice. They also reported that serum 1,25(OH)2D levels decreased within 3 h after a rapid bolus injection of recombinant FGF23, whereas reductions in serum Pi concentrations first appeared at 8–9 h after the injection.11.Shimada T. Hasegawa H. Yamazaki Y. et al.FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.J Bone Miner Res. 2004; 19: 429-435Crossref PubMed Scopus (1243) Google Scholar Furthermore, in patients with tumor-induced osteomalacia, elevated FGF23 levels rapidly decreased after removal of the responsible tumor, and serum levels of Pi and 1,25(OH)2D increased following the decrease in FGF23.12.Weber T.J. Liu S. Indridason O.S. Quarles L.D. Serum FGF23 levels in normal and disordered phosphorus homeostasis.J Bone Miner Res. 2003; 18: 1227-1234Crossref PubMed Scopus (281) Google Scholar, 13.Yamazaki Y. Okazaki R. Shibata M. et al.Increased circulatory level of biologically active full-length FGF-23 in patients with hypophosphatemic rickets/osteomalacia.J Clin Endocrinol Metab. 2002; 87: 4957-4960Crossref PubMed Scopus (534) Google Scholar, 14.Takeuchi Y. Suzuki H. Ogura S. et al.Venous sampling for fibroblast growth factor-23 confirms preoperative diagnosis of tumor-induced osteomalacia.J Clin Endocrinol Metab. 2004; 89: 3979-3982Crossref PubMed Scopus (131) Google Scholar These reports indicate that FGF23 strongly suppresses 1,25(OH)2D production and elicits hypophosphatemia. On the other hand, hyperphosphatemic patients with chronic kidney disease showed significant elevation in circulating FGF23, which correlated with serum Pi and creatinine (CRE) levels,12.Weber T.J. Liu S. Indridason O.S. Quarles L.D. Serum FGF23 levels in normal and disordered phosphorus homeostasis.J Bone Miner Res. 2003; 18: 1227-1234Crossref PubMed Scopus (281) Google Scholar, 15.Larsson T. Nisbeth U. Ljunggren Ö et al.Circulating concentration of FGF-23 increases as renal function declines in patients with chronic kidney disease, but does not change in response to variation in phosphate intake in healthy volunteers.Kidney Int. 2003; 64: 2272-2279Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 16.Imanishi Y. Inaba M. Nakatsuka K. et al.FGF-23 in patients with end-stage renal disease on hemodialysis.Kidney Int. 2004; 65: 1943-1946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar suggesting a possible feedback system in which serum FGF23 regulates Pi in response to serum Pi levels. Saito et al.17.Saito H. Maeda A. Ohtomo S. et al.Circulating FGF-23 is regulated by 1a,25-dihydroxyvitamin D3 and phosphorus in vivo.J Biol Chem. 2005; 280: 2543-2549Crossref PubMed Scopus (355) Google Scholar demonstrated that a dietary Pi loading for 4 weeks led to hyperphosphatemia and increased serum FGF23 levels in 5/6 nephrectomized rats. In normal mice, serum FGF23 levels were regulated by serum Pi controlled by dietary Pi loading for 5 days.18.Perwad F. Azam N. Zhang M.Y. et al.Dietary and serum phosphorus regulate fibroblast growth factor 23 expression and 1,25(OH)2D metabolism in mice.Endocrinology. 2005; 146: 5358-5364Crossref PubMed Scopus (305) Google Scholar Furthermore, Ferrari et al.19.Ferrari S.L. Bonjour J.P. Rizzoli R. Fibroblast growth factor-23 relationship to dietary phosphate and renal phosphate handling in healthy young men.J Clin Endocrinol Metab. 2005; 90: 1519-1524Crossref PubMed Scopus (403) Google Scholar showed that FGF23 was implicated in the physiological regulation of Pi homeostasis independent of PTH, particularly in response to a dietary Pi loading for 5 days in humans. These findings suggest that serum FGF23 levels increase in response to several days of a high Pi diet and/or by continuous elevation of serum Pi levels. Dietary Pi also acutely controls renal Pi reabsorption.1.Berndt T.J. Knox F.G. Renal regulation of phosphate excretion.in: Seldin D.W. Diebish G. The Kidney, Physiology and Pathophysiology. 2nd edn. Raven press, New York1992: 2511-2532Google Scholar,3.Murer H. Hernando N. Forster I. Biber J. Proximal tubular phosphate reabsorption: molecular mechanisms.Physiol Rev. 2000; 80: 1373-1409Crossref PubMed Scopus (424) Google Scholar In rats, Martin et al.20.Martin D.R. Ritter C.S. Slatopolsky E. Brown A.J. Acute regulation of parathyroid hormone by dietary phosphate.Am J Physiol Endocrinol Metab. 2005; 289: E729-E734Crossref PubMed Scopus (64) Google Scholar have documented that oral Pi loading can regulate plasma PTH levels within minutes. Both PTH and FGF23 inhibit proximal tubule Pi reabsorption, resulting in the lowering of serum Pi levels. If FGF23 plays an important role in Pi regulation, FGF23 could be rapidly secreted to adapt to dietary Pi changes. In this study, we investigated the acute response of FGF23 to oral Pi loading in healthy men with an assay system for intact-FGF23 (iFGF23) (Table 1 and Figure 1).Table 1Individual baseline characteristics of subjectsSubjectsABCDEFGHAge (years)2220212120342321Height (cm)172168168176173171169173Weight (kg)64.263.160.064.947.864.957.568.2Serum Ca (mg/dl)9.410.49.09.39.19.39.79.5Serum Pi (mg/dl)3.23.13.83.93.42.73.43.4Serum iPTH (pg/ml)2331202322281822Serum 25(OH)D (ng/ml)3125252527243125Serum 1,25(OH)2D (pg/ml)4183558362697257Serum iFGF23 (pg/ml)3732272640101333Serum creatinine (mg/dl)1.11.11.01.01.10.91.11.0Blood glucose (mg/dl)85856178807679861,25(OH)2D, 1,25-dihydroxyvitamin D; iFGF23, intact-fibroblast growth factor 23; iPTH, intact-parathyroid hormone; Pi, inorganic phosphate. Open table in a new tab 1,25(OH)2D, 1,25-dihydroxyvitamin D; iFGF23, intact-fibroblast growth factor 23; iPTH, intact-parathyroid hormone; Pi, inorganic phosphate. Serum Pi levels increased slightly after ingestion of the meal containing normal amounts of Pi (P400) (Figure 2a). Ingestion of the P800 and P1200 meals prolonged the significantly higher in serum Pi levels from 0.5 h until the end of the experiment compared to that of the P400 meal. Serum Pi levels from 1 to 6 h following the P1200 meal were also significantly higher than those following the P800 meal (Figure 2a). The area under the curve (AUC) for serum Pi calculated over 8 h markedly increased as the intake of Pi increased. The AUCs after P800 (444±44 mg min/ml) and P1200 (519±38 mg min/ml) meals were significantly greater than that after the P400 meal (217±56 mg min/ml) (P=0.024 vs P800, P=0.002 vs P1200). In addition, the postprandial peak values after the P1200 meal exceeded the normal range (2.5–4.5 mg/dl) in seven out of eight subjects. Urinary Pi excretion gradually increased until 6 h after ingestion of the P400 meal, and the excretion after P800 and P1200 meals markedly increased within 1 h compared with that after P400 (Table 2). As shown in Figure 3a, the cumulative urinary Pi excretion was significantly increased in response to Pi loading, the amounts during 8 h after the meal ingestion were 355±26, 455±24, and 605±18 mg for the P400, P800, and P1200 meals, respectively (P400 vs P800 (P=0.0058), P400 vs P1200 (P<0.001), P800 vs P1200 (P=0.001)). On the other hand, the fractional renal tubular reabsorption rate of Pi (% tubular reabsorption of phosphate (%TRP)) was decreased by Pi loading in a dose-dependent manner (Figure 3b). After ingestion of the P1200 meal, the %TRP decreased sharply until 2 h and afterward increased slightly up to 8 h. Serum levels and urinary excretion of CRE were not changed during the experimental period in any subjects (Figure 2c and Table 2).Table 2Postprandial changes in urinary excretionPostprandialBaseline1 h2 h3 h4 h5 h6 h7 h8 hPi (mg) P40013±2.723±3.934±4.0*P<0.01 vs baseline in the same meal.45±4.4*P<0.01 vs baseline in the same meal.46±3.8*P<0.01 vs baseline in the same meal.55±5.1*P<0.01 vs baseline in the same meal.57±3.2*P<0.01 vs baseline in the same meal.50±3.2*P<0.01 vs baseline in the same meal.45±3.3*P<0.01 vs baseline in the same meal. P80011±1.932±3.8*P<0.01 vs baseline in the same meal.59±5.0*P<0.01 vs baseline in the same meal.60±5.6*P<0.01 vs baseline in the same meal.62±4.9*P<0.01 vs baseline in the same meal.70±5.1*P<0.01 vs baseline in the same meal.64±4.5*P<0.01 vs baseline in the same meal.57±4.1*P<0.01 vs baseline in the same meal.51±2.7*P<0.01 vs baseline in the same meal. P120012±2.447±4.1*P<0.01 vs baseline in the same meal.90±4.0*P<0.01 vs baseline in the same meal.90±3.6*P<0.01 vs baseline in the same meal.83±5.1*P<0.01 vs baseline in the same meal.83±4.5*P<0.01 vs baseline in the same meal.77±4.3*P<0.01 vs baseline in the same meal.72±2.7*P<0.01 vs baseline in the same meal.63±1.7*P<0.01 vs baseline in the same meal.Ca (mg) P40010±1.48.3±1.411±1.710±1.49.0±1.26.5±1.14.5±0.83.5±0.7*P<0.01 vs baseline in the same meal.3.5±0.5*P<0.01 vs baseline in the same meal. P80011±1.19.3±0.810±0.98.6±1.27.6±1.25.1±0.9*P<0.01 vs baseline in the same meal.3.7±0.5*P<0.01 vs baseline in the same meal.2.8±0.5*P<0.01 vs baseline in the same meal.2.3±0.4*P<0.01 vs baseline in the same meal. P120010±1.38.4±1.08.4±1.06.9±1.16.5±1.25.0±1.0*P<0.01 vs baseline in the same meal.3.3±0.6*P<0.01 vs baseline in the same meal.2.2±0.3*P<0.01 vs baseline in the same meal.2.0±0.3*P<0.01 vs baseline in the same meal.CRE (mg) P40065±2.666±3.672±3.076±5.371±4.171±3.769±3.267±3.867±4.0 P80066±3.668±4.073±3.773±3.273±3.470±2.768±2.768±3.366±3.7 P120064±3.067±3.872±3.573±3.270±3.571±2.768±2.669±2.767±3.9Ca, calcium; CRE, creatinine; Pi, inorganic phosphate.Values are mean±s.e.m. for eight subjects.The baseline values were calculated by dividing the urinary excretion volume collected from 0800 to 1200 hours by 4.* P<0.01 vs baseline in the same meal. Open table in a new tab Ca, calcium; CRE, creatinine; Pi, inorganic phosphate. Values are mean±s.e.m. for eight subjects. The baseline values were calculated by dividing the urinary excretion volume collected from 0800 to 1200 hours by 4. Serum Ca levels did not change as the intake of Pi increased (Figure 2b). As shown in Figure 4a, there were some significance in the levels of serum 1,25(OH)2D between different Pi loaded groups, but no dependency on the intake of Pi amount was observed in serum 1,25(OH)2D levels. Serum intact-PTH (iPTH) levels at 1, 2, and 4 h after the P800 and P1200 meals significantly increased compared with those after the P400 meal (Figure 4b). Time-dependent changes of serum iPTH levels demonstrated a dual-phase curve as the intake of Pi increased. The first peak was observed at 1 h after the meal ingestion, and the second peak at 6 h. The peak values of iPTH after P1200 were 128±11 and 141±15% compared with baseline, and at 6 h were also significantly higher than P400 (P=0.016). Urinary Ca excretion decreased gradually and significantly compared with baseline values collected from 0800 hours to noon in all test meals (Table 2). There was no significant change in urinary Ca excretion in response to Pi loading in the comparison of different meals. The cumulative Ca excretion amounts during 8 h after the meal ingestion were 55.7±8.5 mg (P400), 49.4±5.3 mg (P800), and 42.7±5.8 mg (P1200). Baseline levels of serum iFGF23 in eight subjects varied within the range of 9–42 pg/ml, but the individual baseline values at each experimental day were similar (Figure 5a). As shown in Figure 5b, serum iFGF23 levels after the ingestion of P400 meal were gradually decreased over the period. Interestingly, serum iFGF23 levels in the P800 meal were significantly lower than those in the P400 meal from 1 to 6 h after the ingestion (P<0.05). After the ingestion of P1200 meal, serum iFGF23 levels were not changed up to 6 h, but significantly increased at 8 h compared with P400 and P800 meals (P<0.001). To examine within-individual associations between levels of the iFGF23 and iPTH, and measures of serum Pi levels and %TRP, Spearman's non-parametric correlation coefficients (rs) were estimated after centralizing biomarker variables by their median within each individual (see Materials and Methods section). As shown in Figure 6a and b, significant negative association was detected between iFGF23 and serum Pi levels (rs=-0.21, P=0.002), whereas positive association was observed between iFGF23 and renal Pi reabsorption rate (%TRP) (rs=0.18, P=0.007). We also carried out similar analyses, resulting that iPTH levels was significantly associated with both serum Pi levels (rs=0.35, P<0.001) and %TRP (rs=-0.34, P<0.001) (Figure 6c and d). In the present study, we investigated an acute effect of Pi loading on serum iFGF23 levels in healthy men with postprandial time-course experiments. We hypothesized that FGF23 would rapidly respond to Pi loading. However, the present data demonstrated that serum iFGF23 levels were decreased or not changed up to 6 h in all diets, but modestly increased at 8 h after the highest loading (1200 mg). Interestingly, serum iFGF23 levels after P800 meal were significantly lower than that of P400 meal. We do not know the reason, but the data indicated that serum iFGF23 did not increased in response to Pi loading within 6 h. Additionally, serum iFGF23 showed negative correlation with serum Pi level, but positive correlation with %TRP in this short period experiment. FGF23 is a phosphaturic factor that strongly decreases serum Pi level via inhibition of renal Pi reabsorption.4.Shimada T. Mizutani S. Muto T. et al.Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia.Proc Natl Acad Sci USA. 2001; 98: 6500-6505Crossref PubMed Scopus (1120) Google Scholar, 9.Yu X. White K.E. FGF23 and disorders of phosphate homeostasis.Cytokine Growth Factor Rev. 2005; 16: 221-232Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10.Shimada T. Muto T. Urakawa I. et al.Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo.Endocrinology. 2002; 143: 3179-3182Crossref PubMed Scopus (354) Google Scholar Therefore, there is the discrepancy between the FGF23 action and relationship between serum iFGF23 levels and %TRP. On the other hand, we observed significant and reasonable association of serum iPTH levels with both serum Pi levels and %TRP. These results suggest that PTH can rapidly respond to dietary Pi loading but not FGF23. Larsson et al.15.Larsson T. Nisbeth U. Ljunggren Ö et al.Circulating concentration of FGF-23 increases as renal function declines in patients with chronic kidney disease, but does not change in response to variation in phosphate intake in healthy volunteers.Kidney Int. 2003; 64: 2272-2279Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar reported that FGF23 levels did not change after Pi deprivation or Pi loading. However, they noted that high levels of FGF23 were found in four of six volunteers on the high Pi intake. They collected blood samples from their subjects four times per day at 4 h intervals, and demonstrated that administration of high doses of Pi failed to trigger a rise in FGF23, despite significant increases in renal Pi clearance. Therefore, they suggested that FGF23 is not responsible for the acute adjustment of Pi excretion in the kidney. Ferrari et al.19.Ferrari S.L. Bonjour J.P. Rizzoli R. Fibroblast growth factor-23 relationship to dietary phosphate and renal phosphate handling in healthy young men.J Clin Endocrinol Metab. 2005; 90: 1519-1524Crossref PubMed Scopus (403) Google Scholar demonstrated that Pi loading for 3 days increased serum FGF23 levels in the morning after fasting in spite of normal serum Pi levels. They also demonstrated that changes in FGF23 were positively correlated with changes in 24 h urinary Pi excretion, and negatively correlated with changes in the maximal Pi reabsorption, but PTH was not. Additionally, serum FGF23 levels were not correlated with serum Pi levels in the morning after fasting. Considering these data, Pi loading was probably involved in the postprandial increase in serum Pi levels, resulting in increases in serum FGF23, which reduced serum Pi levels overnight before the morning sampling. Furthermore, they measured both intact and C-terminal degradation products of FGF23. In the morning after fasting, iFGF23 might revert to normal levels, but C-terminal degradation products might remain elevated. Therefore, the data may reflect the chronic adaptation of Pi homeostasis through FGF23 action. Previous studies demonstrated that dietary Pi loading increased serum FGF23 levels.17.Saito H. Maeda A. Ohtomo S. et al.Circulating FGF-23 is regulated by 1a,25-dihydroxyvitamin D3 and phosphorus in vivo.J Biol Chem. 2005; 280: 2543-2549Crossref PubMed Scopus (355) Google Scholar, 18.Perwad F. Azam N. Zhang M.Y. et al.Dietary and serum phosphorus regulate fibroblast growth factor 23 expression and 1,25(OH)2D metabolism in mice.Endocrinology. 2005; 146: 5358-5364Crossref PubMed Scopus (305) Google Scholar, 19.Ferrari S.L. Bonjour J.P. Rizzoli R. Fibroblast growth factor-23 relationship to dietary phosphate and renal phosphate handling in healthy young men.J Clin Endocrinol Metab. 2005; 90: 1519-1524Crossref PubMed Scopus (403) Google Scholar, 21.Shimada T. Yamazaki Y. Takahashi M. et al.Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism.Am J Physiol Renal Physiol. 2005; 289: F1088-F1095Crossref PubMed Scopus (267) Google Scholar What is necessary to increase serum FGF23 level in response to Pi loading? One possible explanation is that long-term hyperphosphatemia, as is often the case in patients with chronic kidney disease, caused the increase in FGF23 production. Recently, Perwad et al.18.Perwad F. Azam N. Zhang M.Y. et al.Dietary and serum phosphorus regulate fibroblast growth factor 23 expression and 1,25(OH)2D metabolism in mice.Endocrinology. 2005; 146: 5358-5364Crossref PubMed Scopus (305) Google Scholar suggested that dietary Pi loading for 5 days upregulated serum FGF23 levels in mice. Saito et al.17.Saito H. Maeda A. Ohtomo S. et al.Circulating FGF-23 is regulated by 1a,25-dihydroxyvitamin D3 and phosphorus in vivo.J Biol Chem. 2005; 280: 2543-2549Crossref PubMed Scopus (355) Google Scholar demonstrated that ingestion of high Pi diet for 4 weeks increased both serum Pi and FGF23 level in 5/6 nephrectomized rats. More recent studies also demonstrated that Pi loading increased serum iFGF23 levels, whereas Pi depletion decreased serum iFGF23 in human.22.Antoniucci D.M. Yamashita T. Portale A.A. Dietary phosphorus regulates serum fibroblast growth factor-23 concentrations in healthy men.J Clin Endocrinol Metab. 2006; 91: 3144-3149Crossref PubMed Scopus (332) Google Scholar,23.Burnett S.A. Gunawardene S.C. Bringhurst F.R. et al.Regulation of C-terminal and intact FGF-23 by dietary phosphate in men and women.J Bone Miner Res. 2006; 21: 1187-1196Crossref PubMed Scopus (351) Google Scholar These observations suggest that long-term dietary Pi loading and long-term hyperphosphatemia would be important regulator under physiological and pathological conditions. Burnett et al.,23.Burnett S.A. Gunawardene S.C. Bringhurst F.R. et al.Regulation of C-terminal and intact FGF-23 by dietary phosphate in men and women.J Bone Miner Res. 2006; 21: 1187-1196Crossref PubMed Scopus (351) Google Scholar observed the elevation of serum iFGF23 levels more than 1 day after beginning of the Pi loading (2500 mg diet per day), but they did not estimate it less than 1 day. On the other hand, our data suggest that iFGF23 levels cannot rapidly increase in response to the Pi loading, but increased at 8 h after the ingestion of P1200 diet. This difference would be due to the difference of observed period. As far as we know, there is no report to investigate rapid regulation of FGF23. It is technically very difficult to carry out the balance study in rats and mice, especially in short period, because there are some limitations to correct urine and serum samples every 1 h in such animals. Thus, this is the first report to investigate the rapid regulation of FGF23 in response to dietary Pi in detail. In this study, we hypothesized that the rapid regulation of Pi homeostasis may be mediated by FGF23 in part. As described in previous reports,24.Reiss E. Canterbury J.M. Bercovitz M.A. Kaplan E.L. The role of phosphate in the secretion of parathyroid hormone in man.J Clin Invest. 1970; 49: 2146-2149Crossref PubMed Scopus (168) Google Scholar, 25.Calvo M.S. Heath III, H. Acute effects of oral phosphate-salt ingestion on serum phosphorus, serum ionized calcium, and parathyroid hormone in young adults.Am J Clin Nutr. 1988; 47: 1025-1029PubMed Google Scholar, 26.Portale A.A. Halloran B.P. Morris Jr, R.C. Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D.J Clin Invest. 1987; 80: 1147-1154Crossref PubMed Scopus (179) Google Scholar our results also revealed that dietary Pi intake acutely decreased renal phosphate reabsorption (%TRP), and increased serum PTH levels within 1 h after Pi loading. However, there was only one time point at 8 h in individuals fed a P1200 meal where iFGF23 was modestly increased. Despite of the modest increase in iFGF23 at 6–8 h after Pi loading, tubular Pi reabsorption slightly increased rather than decreased during that time. Additionally, opposite correlation between serum iFGF23 and %TRP was observed. Syal et al.27.Syal A. Schiavi S. Chakravarty S. et al.Fibroblast growth factor-23 increases mouse PGE2 production in vivo and in vitro.Am J Physiol Renal Physiol. 2006; 290: F450-F455Crossref PubMed Scopus (15) Google Scholar reported that FGF23 increased prostaglandin E2 (PGE2) production in renal tubular cells to inhibit sodium-dependent Pi transport activity. This would be an important pathway for the FGF23-mediated signal. They also demonstrated that FGF23 required 6 h to increase PGE2 production in vivo. Shimada et al.11.Shimada T. Hasegawa H. Yamazaki Y. et al.FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.J Bone Miner Res. 2004; 19: 429-435Crossref PubMed Scopus (1243) Google Scholar also found that an increase in fractional excretion of Pi and lower serum Pi level were observed 9 h after the intravenous injection of FGF23 in vivo. Therefore, the time-lag of FGF23 action in vivo and the unparallel changes of serum Pi and iFGF23 we described above may be involved in the complicated changes in serum iFGF23 levels after dietary Pi loading within short period. Unlike FGF23, PTH rapidly, about 1 h, responded to Pi loading as described previously.24.Reiss E. Canterbury J.M. Bercovitz M.A. Kaplan E.L. The role of phosphate in the secretion of parathyroid hormone in man.J Clin Invest. 1970; 49: 2146-2149Crossref PubMed Scopus (168) Google Scholar,25.Calvo M.S. Heath III, H. Acute effects of oral phosphate-salt ingestion on serum phosphorus, serum ionized calcium, and parathyroid hormone in young adults.Am J Clin Nutr. 1988; 47: 1025-1029PubMed Google Scholar Our data have demonstrated the double peak response of serum PTH levels to dietary Pi loading. Recently, Martin et al.20.Martin D.R. Ritter C.S. Slatopolsky E. Brown A.J. Acute regulation of parathyroid hormone by dietary phosphate.Am J Physiol Endocrinol Metab. 2005; 289: E729-E734Crossref PubMed Scopus (64) Google Scholar have demonstrated that immediate effect of Pi on PTH would not be dependent on serum Pi increase, suggesting that additional signal arising from gastrointestinal tract and serum Pi would contribute to the rapid response. These factors may also mediate the first peak of serum PTH increase in our result. On the other hand, the second peak was probably mediated by serum Pi increase after dietary Pi loading. In the previous experiment, the subjects were administered high Pi diet with high Ca to minimize the response of PTH to Pi loading.19.Ferrari S.L. Bonjour J.P. Rizzoli R. Fibroblast growth factor-23 relationship to dietary phosphate and renal phosphate handling in healthy young men.J Clin Endocrinol Metab. 2005; 90: 1519-1524Crossref PubMed Scopus (403) Google Scholar Recently, Shimada et al.21.Shimada T. Yamazaki Y. Takahashi M. et al.Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism.Am J Physiol Renal Physiol. 2005; 289: F1088-F1095Crossref PubMed Scopus (267) Google Scholar have suggested that Ca is one of the regulatory factors of FGF23 production in mice. Therefore, high Ca intake could be required for the induction of FGF23 in response to Pi loading. In our study, to examine the regulation of FGF23 under a standard Ca intake in Japanese, subjects received a diet containing Ca standardized to the Japanese recommended daily allowance (about 200 mg per meal) throughout the study. Therefore, the Pi:Ca ratio in the experimental meal was 2:1 in P400, 4:1 in P800, 6:1 in P1200. Ca intake was therefore relatively lower than that in previous works.19.Ferrari S.L. Bonjour J.P. Rizzoli R. Fibroblast growth factor-23 relationship to dietary phosphate and renal phosphate handling in healthy young men.J Clin Endocrinol Metab. 2005; 90: 1519-1524Crossref PubMed Scopus (403) Google Scholar, 22.Antoniucci D.M. Yamashita T. Portale A.A. Dietary phosphorus regulates serum fibroblast growth factor-23 concentrations in healthy men.J Clin Endocrinol Metab. 2006; 91: 3144-3149Crossref PubMed Scopus (332) Google Scholar, 23.Burnett S.A. Gunawardene S.C. Bringhurst F.R. et al.Regulation of C-terminal and intact FGF-23 by dietary phosphate in men and women.J Bone Miner Res. 2006; 21: 1187-1196Crossref PubMed Scopus (351) Google Scholar Under these conditions, serum iFGF23 did not remarkably responded to Pi loading in the present study. We cannot conclude the role of Ca in the regulation of FGF23, but this study may reflect the physiological FGF23 response to Pi loading with a standard Japanese diet. Additionally, there is also no report about diurnal course of FGF23, therefore, the time-course of P400 would reflect, in part, real diurnal changes from noon to 2000 hours in our daily life. So we think that all data in additional Pi loading (P800 and P1200 meals) should be compared with those in P400 meal. In summary, it has been reported that FGF23 plays an important role in the phosphate homeostasis. However, our study demonstrated that serum FGF23 level did not rapidly increase in response to dietary Pi loading, whereas PTH did. Our results suggest that FGF23 seemingly has no bearing on the rapid regulation of Pi homeostasis more than we expected, although FGF23 probably is a phosphaturic factor as reported by many papers. There is still more to learn about the roles of FGF23 and PTH in the regulation of phosphate homeostasis. Eight healthy male volunteers between 20 and 34 years of age without apparent health problems or medication use were recruited for this study. The study protocols were approved by the Ethics Committee of the Tokushima University Hospital. Signed written informed consent was obtained from each study subject before participation. The baseline characteristics of the eight subjects are shown in Table 1. The protocol utilized in the clearance studies is illustrated diagrammatically in Figure 1. The study used a randomized, double-blind, crossover design on 3 different days separated by at least 7 days. The subjects were alternately served one of three test meals containing different Pi amounts adjusted by Pi supplement as lunch at noon. The test meals were as follows: (1) P400, standard meal containing 400 mg Pi and placebo supplement; (2) P800, standard meal and a 400 mg Pi supplement; and (3) P1200, standard meal and a 800 mg Pi supplement. The standard meal consisted of steamed rice, boiled egg, ham, and milk (690 kcal, 110 g carbohydrate, 23 g protein, 16 g fat, 400 mg Pi, and 200 mg Ca). Pi supplement was administered orally as a solution of neutral sodium phosphate (mixture of Na2HPO4 and NaH2PO4). To keep the intakes of Na constant among three test meals, the amount of Na in each placebo and Pi supplement was adjusted to 1022 mg by NaCl (for P400, placebo supplement contained 1022 mg Na as NaCl; for P800, 400 mg Pi supplement contained 511 mg Na in NaCl and 511 mg Na in sodium phosphate; for P1200, 800 mg Pi supplement contained 1022 mg Na in sodium phosphate). The subjects drank each supplement dissolved in 22 ml water in the same time during a meal. All of the meals were consumed over 7–14 min. The subjects remained sedentary during the experimental period and drank the appointed water (100 ml/h) only. Before each study day, the subjects were asked to abstain from foods and beverages other than water, not containing Pi, after 1400 hours. They were served a standard dinner (650 kcal, 100 g carbohydrate, 23 g protein, 16 g fat, 480 mg Pi, and 250 mg Ca) at 2000 hours, and instructed to go to bed at 2400 hours. On each study day, the subjects consumed a standard breakfast (620 kcal, 110 g carbohydrate, 22 g protein, 14 g fat, 330 mg Pi, and 200 mg Ca) after urination at 0800 hours. Urine was collected from 0800 to 1200 hours, as a baseline sample. Blood samples were collected immediately before (0 h; baseline) and at 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h after test meal ingestion. Venous blood was taken from an antecubital vein for the measurement of serum Pi, Ca, CRE, 1,25(OH)2D, iPTH, and iFGF23 concentrations. Aliquots of serum were stored at -80°C until assayed. Urine samples after test meal ingestion were also collected every hour from noon to 2000 hours, and volumes were recorded. All urine samples were stored at -30°C until assayed. We measured urinary concentrations of Pi, Ca, and CRE. Serum and urinary concentrations of Pi, Ca, and CRE were measured using each specific assay kit (Wako Pure Chemical Industries Ltd, Osaka, Japan). Serum 25-hydroxyvitamin D, 1,25(OH)2D, and iPTH were analyzed by MBC (Mitsubishi Kagaku Bio-Clinical Laboratories Inc., Tokyo, Japan) using a radioimmunoassay and an immunoradiometric assay, respectively. The fractional renal tubular reabsorption rate of Pi (%TRP) was calculated from the equation %TRP=100 × (1-(urine Pi concentration × serum CRE concentration/urine CRE concentration × serum Pi concentration)). FGF23 can be processed by proteases to release a small C-terminal peptide. The C-terminal proteolytic peptide may complicate the evaluation of iFGF23.28.Ito N. Fukumoto S. Takeuchi Y. et al.Comparison of two assays for fibroblast growth factor (FGF)-23.J Bone Miner Metab. 2005; 23: 435-440Crossref PubMed Scopus (63) Google Scholar Recent publication by Burnett et al.23.Burnett S.A. Gunawardene S.C. Bringhurst F.R. et al.Regulation of C-terminal and intact FGF-23 by dietary phosphate in men and women.J Bone Miner Res. 2006; 21: 1187-1196Crossref PubMed Scopus (351) Google Scholar pointed out that iFGF23 can more precisely reflect physiological response than C-terminal FGF23. Therefore, we have used intact FGF23 kit (Kainos Laboratories Inc., Tokyo, Japan) for this investigation and this is reasonable for the evaluation of FGF23 status in our experiment. To examine the time-course behaviors, we performed mixed model regression analysis to consider repeated measures of crossover setting. In the regression analyses, one of serum and urinary biomarkers was included as a dependent variable; and time, Pi loading levels, time–Pi interaction were included as independent variables. We considered it appropriate for our analyses to use Pi loading level as a categorical variable, resulted from likelihood ratio tests comparing regression models with Pi loading levels treated as continuous or categorical variable. Therefore, mainly Pi loading levels were treated as categorical. With respect to assumptions of regression analysis and goodness-to-fit by examination of distribution normality, Akaike Information Criteria, and properties of residuals from mixed model, we decided to use the data on log-transformed relative changes in levels of serum iFGF23, serum 1,25-(OH)2D, and serum PTH, the data on absolute changes in levels of %TRP, and the crude data on levels of serum calcium, serum Pi, and serum CRE. When results derived from log-transformed data, geometric means and their standard errors were presented. In the analyses with dependent variables representing relative or absolute changes, the effect of statistical adjustment for baseline value of outcome measures was considered to account for the differences in baseline values. Results with and without the adjustment were little different (data not shown). Therefore, in all regression models, we included the variable representing the baseline values to avoid potential bias due to variability of baseline values. To examine associations between levels of iFGF23 and PTH, and measures of serum Pi levels and %TRP, Spearman's non-parametric correlation coefficients were estimated. Non-parametric procedure was selected to avert requirements of normal distributions and linear associations of variables of interests. To quantify the associations between biomarkers in acute response to Pi loading within each individual, estimated correlations need to be independent of between-individual variability. Hence, we centralized each biomarker variable within each individual by median value so that correlation statistics were independent of between-individual variability. Here, results of Spearman's correlation coefficients from data median-centralized within each individual are shown in this paper. We also applied one-way analysis of variance to compare values for the AUC for serum Pi calculated over 8 h (AUC comparison) and cumulative urinary Pi excretion with consequence post hoc Fisher's PLSD comparisons for the AUC comparison. In our analyses, two-sided P-values were estimated. We regarded P-value less than 0.05 statistically significant. Statistical analyses were performed by SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (YT), for Young Scientists (YT, HO, HA, and HY), and for Exploratory Research (KN and ET) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan, and was also supported by Uehara Memorial Foundation (YT), and the 21st Century COE Program, Human Nutritional Science and Stress Control, Tokushima, Japan (ET).
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