Chinese Medical Journal 2010;123(9):1158-1162
Levels and dynamic changes of serum fibroblast growth factor 23 in hypophosphatemic rickets/osteomalacia

XIA Wei-bo,  JIANG Yan,  LI Mei,  XING Xiao-ping,  WANG Ou,  HU Ying-ying,  ZHANG Hua-bing,  LIU Huai-cheng,  MENG Xun-wu,  ZHOU Xue-ying

XIA Wei-bo (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

JIANG Yan (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

LI Mei (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

XING Xiao-ping (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

WANG Ou (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

HU Ying-ying (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

ZHANG Hua-bing (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

LIU Huai-cheng (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

MENG Xun-wu (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

ZHOU Xue-ying (Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China)

Correspondence to:XIA Wei-bo,Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China (Tel: 86-10-6529-5358. Fax:86-10-6529-5358. E-mail:xiaweibo@medmail.com.cn)
Keywords
fibroblast growth factor 23; hypophosphatemia; rickets; osteomalacia
Abstract

Background  Hypophosphatemic rickets/osteomalacia is a group of diseases characterised by defective mineralization of bone due to hypophosphatemia and low 1,25-dihydroxy vitamin D. To explore the role of fibroblast growth factor 23 (FGF-23) in the regulation of phosphate homeostasis, we measured the circulating concentrations of this growth factor in healthy individuals and in patients with hypophosphatemic rickets/osteomalacia.
Methods  Nineteen patients with hypophosphatemic rickets/osteomalacia were included in hypophosphatemic group (HP, 12 female and 7 male, mean age was 30 years), and 19 healthy age-matched individuals served as the control group. Full length FGF-23 fragments were measured by two-site enzyme-linked immunosorbent assay.
Results  Mean FGF-23 concentrations were significantly higher in the HP group ((87.4 ± 43.6) pg/ml) compared with the control group ((19.2 ± 6.16) pg/ml; P <0.001). In 1 patient with tumour-induced osteomalacia, serum FGF-23 concentrations were 84.1 pg/ml; these concentrations were normalized 2 hours after a hemangiopericytoma resection (7.8 pg/ml). Subsequently, serum 1,25(OH)2 vitamin D3 concentrations significantly increased from 21.3 pg/ml to 89.3 pg/ml, and serum phosphorus levels were normalized.
Conclusions  Serum FGF-23 concentrations were markedly elevated in patients with hypophosphatemic rickets. FGF-23 plays an important role in the pathogenesis of hypophosphatemic rickets/osteomalacia.

Hypophosphatemic rickets/osteomalacia (or hypo- phosphatemic vitamin D-resistant rickets/ osteomalacia) is a group of diseases characterised by defective mineralization of bone due to hypophos- phatemia and low 1,25-dihydroxy vitamin D. There are four main subclinical types of this disorder: X-linked hypophosphatemic rickets (XLH), autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemia (ARHP) and tumour-induced osteomalacia (TIO).1 In these disorders, hypophos- phatemia is caused by circulating factors, such as phosphatonins, which are responsible for renal phosphate-wasting. Fibroblast growth factor 23 (FGF-23) is one of the most important candidate phosphatonins.2 To explore the pathophysiologic role of FGF-23 in hypophosphatemic rickets/osteomalacia, we measured the circulating concentrations of this growth factor in healthy individuals and in patients with hypophosphatemic rickets/osteomalacia.

METHODS

Subjects
Nineteen patients (12 female and 7 male) with hypophosphatemic rickets/osteomalacia were included in the hypophosphatemic group (HP). Mean age of patients with HP was 30 (5–63) years. Inclusion criteria of HP were hypophosphatemia associated with renal phosphate wasting, bone deformities and radiological signs of rickets or osteomalacia. Exclusion criteria were the presence of tubulopathy, hypercalciuria, or hyperpara- thyroidism before the onset of treatment. Nineteen healthy age-matched individuals (12 female and 7 female) were enrolled in the control group (Ctrl). In the HP group, one 37-year-old male had TIO; his hypophos- phatemia and osteomalacia were corrected following tumor resection. The lesion was confirmed surgically, and pathology demonstrated hemangiopericytomas. Serum samples were obtained for measurement before, as well as at 2, 4, 6, 12, 24, 48, 72 hours and 5 days after tumor removal. Four patients from two families were diagnosed with XLH by Phex gene mutation detection.3 One 7- year-old female patient had McCune Albright’s syndrome (MAS) with hypophosphatemic rickets. Her serum phosphorus was lower than normal. Radiographs revealed generalized cortical thinning and uniform trabecular resorption, enlargement of epiphyses in some metacarpal bones, and distal metaphysis of long bones. All of the remaining 13 patients had sporadic hypophosphatemic rickets or osteomalacia. All 13 patients had negative family history. No mutation was found in Phex, DMP1 and FGF-23 gene in leukocyte DNA. Urinary tests showed negative glucose and amino acid, which excluded Fanconi syndrome.

General characteristics and biochemical parameters of these patients are presented in Table.


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Table. General characteristics and laboratory findings for patients with hypophosphatemic rickets /osteomalacia

This study was approved by Department of Scientific Research of Peking Union Medical College Hospital. Informed consent was obtained prior to collection of clinical data and blood samples.

Methods
All laboratory data were collected prior to phosphorus and 1α-hydroxy vitamin D3 or 1,25-dihydroxy vitamin D3 treatment. Patients who received treatment with these medications were asked to wait for 1 week before sample collection. Blood samples were collected after overnight fast, and all the measurements were made from the same blood-sample collection.

Full length FGF-23 fragments were measured by two-site enzyme-linked immunosorbent assay using FGF-23 ELISA Kit (Kainos Co, Japan). Serum 1,25(OH)2 vitamin D was measured using commercially available RIA-CT kits, according to the manufacturer’s instructions (BioSource Europe, S.A. Belgium). Serum intact parathyroid hormone concentration was measured using a solid-phase two-site chemiluminescent immunoassay (Diagnostic Products Corporation, DPC, Tianjin, China). Serum calcium and phosphate levels and other biochemical parameters were measured spectro- photometrically using routine assays available at the central laboratory of Peking Union Medical College Hospital, Beijing, China. Renal tubular maximum phosphate/glomerular filtration rate was calculated from a normogram by Walton and Bijvoet.4

Statistical analyses
Statistical analyses were performed using the SPSS 13.0 software package. Values are expressed as means ± standard deviation (SD). Differences between groups were calculated using Student’s independent t test. A probability of P <0.05 was considered statistically significant.

RESULTS

Serum FGF-23 concentrations
Serum FGF-23 concentration was significantly higher in the HP group ((87.4 ± 43.6) pg/ml) than in the control group ((19.1 ± 6.4) pg/ml, P <0.001) (Figure 1). Serum concentrations of FGF-23 in patients with TIO, XLH and McCune Albright’s syndrome combined with hypo- phosphatemic rickets and sporadic HP are presented in Figure 2. There was no significant difference between patients with XLH and sporadic HP ((84.7±66.6) pg/ml vs. (86.4±40.9) pg/ml, P =0.95). There was no significant difference in serum FGF-23 levels in the HP group prior to treatment with phosphate and active vitamin D and 1 week after discontinuation of treatment ((88.3±45.9) pg/ml (n=15) vs. (84.1±39.5) pg/ml (n=4), P =0.86).


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Figure 1. Serum FGF-23 in patients with hypophosphatemic rickets/osteomalacia (HP) versus age-matched controls (Ctrl).


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Figure 2. Serum FGF-23 in hypophosphatemic rickets/ osteomalacia subgroups versus age-matched controls (Ctrl). MAS: McCune Albright’s syndrome; TIO: tumour-induced osteomalacia; XLH: X-linked hypophosphatemic rickets.

Serum 1,25(OH)2 vitamin D3 concentrations
Serum 1,25(OH)2 vitamin D3 concentrations in 19 patients with hypophosphatemic rickets/osteomalacia were significantly lower than those in the control group ((18.8±7.8) pg/ml vs. (25.4±6.3) pg/ml; P <0.001) (Figure 3).


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Figure 3. Serum 1,25(OH)2 vitamin D3 in patients with hypophosphatemic rickets/osteomalacia (HP) versus age-matched controls (Ctrl).

The dynamic changes of serum FGF-23, 1,25(OH)2 vitamin D3 and phosphorus levels in TIO
Serum FGF-23 concentrations in the patient with TIO were 84.1 pg/ml; this was normalized 2 hours after a hemangiopericytoma resection below his right waist (7.8 pg/ml; Figure 4). It was maintained at this low level until 5 days after tumor resection. Subsequently, levels of serum 1,25(OH)2 vitamin D3 were increased from 21.3 pg/ml to 80.5 pg/ml, and serum phosphorus also increased to normal range 48 hours after tumor resection (Figure 4).


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Figure 4. Dynamic changes in serum FGF-23, 1,25(OH)2 vitamin D3 and serum phosphate in a patient with tumour- induced osteomalacia.

DISCUSSION

The mineralization of cartilage and bone matrix requires an adequate supply of calcium and phosphate. Chronic inorganic phosphate (Pi) insufficiency results in impaired bone mineralization, rickets and osteomalacia.5 Hypophosphatemic disorders include XLH, ADHR, ARHP and TIO, all of which are characterized by renal phosphate wasting and reduced serum 1α, 25 dihydroxy vitamin D concentrations. It has been demonstrated that some circulating factors are responsible for hypophosphatemia and renal phosphate wasting in these disorders. Several proteins, such as FGF-23, a novel phosphate-regulating factor, secreted frizzled-related protein (sFRP-4), matrix extracellular phosphor- glycoprotein (MEPE) and fibroblast growth factor-7 (FGF-7) have been identified as potential circulating factors, known as phosphatonins.6 Among these phosphatonins, FGF-23 may represent the crossroad of inherited and acquired metabolic derangements.7 Therefore, we evaluated serum FGF-23 concentrations in 19 HP patients and 19 control subjects. In addition, we observed the dynamic changes in serum FGF-23 levels in a TIO patient before and after tumor resection.

Recent studies have reported that serum FGF-23 increased in some patients with XLH, and serum phosphorus concentrations were negatively correlated with circulating FGF-23 levels in those patients.8,9 The current study showed that serum FGF-23 levels were significantly higher in patients with hypophosphatemic rickets/osteomalacia compared with healthy controls. No significant differences of serum FGF-23 levels were found in patients with XLH, TIO, sporadic HP or McCune Albright’s syndrome with hypophosphatemic rickets, which indicates that the elevation of serum FGF-23 is a common feature and plays an important and similar pathophysiological role in these hypophosphatemic disorders. This elevation may also induce hypophosphatemia by inhibiting both renal and intestinal Pi absorption through suppression of NaPi-IIa and -IIb production.10-13

Normally, hypophosphatemia strongly stimulates renal 1α-hydroxylase, leading to a rise in the serum concentration of 1,25(OH)2D3.14 However, patients with hypophosphatemic rickets/osteomalacia generally have normal serum concentrations of PTH and calcium in association with low or inappropriately normal levels of circulating 1,25(OH)2 vitamin D3, which counters hypophosphatemia-mediated increases in the renal production of 1,25(OH)2 vitamin D3. The current study showed that serum 1,25(OH)2 vitamin D3 levels in 19 patients with hypophosphatemic rickets/osteomalacia were lower than in the control group, probably due to dysregulation of vitamin D metabolism in the presence of high circulating levels of FGF-23.

Several animal studies have also demonstrated that FGF-23 plays a role in vitamin D metabolism.13,15,16 The FGF23(-/-) mice displayed an elevation of serum 1,25(OH)2 vitamin D3 for the enhanced expression of renal 25-hydroxy vitamin D-1alpha-hydroxylase (1alpha-OHase) at 10 days of age. FGF-23 transgenic mice clearly have increased expression of 24-hydroxylase mRNA in kidneys,8 which contributes to an inability to increase serum 1,25(OH)2 vitamin D3 levels in response to hypophosphatemia. Although relationships between serum FGF-23 levels and 1,25(OH)2 vitamin D3 or serum phosphorus were observed in our study, we could not find any significant correlation. It may be that within the spectrum of hypophosphatemic rickets/osteomalacia there is significant variability in the threshold at which serum FGF-23 levels cause hypophosphatemia and low 1,25(OH)2 vitamin D3 levels.

Some patients with TIO have high levels of FGF-23, which are normalized after tumor resection.9,17-19 We observed dynamic changes in serum FGF-23 concentrations in 1 patient before and after tumor resection. Serum FGF-23 markedly decreased within 2 hours of removal of a hemangiopericytoma, and remained at this low level for a period of 5 days.

Only one study has previously demonstrated the half-life of FGF-23 in humans.20 The current study clearly showed that the cleavage time of FGF-23 in humans was no more than 2 hours. According to these results, serum 1,25(OH)2 vitamin D3 levels notably increased within 24 hours of the removal of the tumor (21.8 pg/ml to 32.1 pg/ml) and this increase continued 72 hours after the operation (80.5 pg/ml), even without any active vitamin D treatment. The rapid reduction of FGF-23 might correct the phosphate wasting and reduce 1,25(OH)2 vitamin D3 production in TIO patients. Serum phosphorus returned to normal within 48 hours of the operation, later than FGF-23 and 1,25(OH)2 vitamin D3. The dynamic changes of serum FGF-23, 1,25(OH)2 vitamin D3, and phosphorus probably provide us with a profile about the pathophysiology of hypophosphatemia in patients with TIO. Excessive FGF-23 secretion from the tumor may inhibit 1,25(OH)2 vitamin D3 production and lead to renal phosphate wasting and impaired intestinal phosphate absorption.

Rickets and osteomalacia in MAS has been shown to be associated with hypophosphatemia and renal phosphate wasting. It has been suggested that renal phosphate wasting in MAS may be caused by either renal tubule cells bearing GSα mutations, or alternatively, by the presence of a circulating phosphaturic factor produced by the dysplastic bone.21 The level of expression of FGF-23 in osteoblast/osteocytes in woven bone in fibrodysplasia tissue was significantly greater in patients with fibrous dysplasia (FD) and HP than FD patients without HP, and nearly equal to that in TIO.22 The serum level of FGF-23 in our patient with MAS and HP was 115 ng/ml, which was higher than the normal range. These results indicate that FGF-23 secreted from dysplastic bone may cause hypophosphatemia in patients with MAS.23

In conclusion, serum FGF-23 concentration is markedly increased in patients with HP. High FGF-23 concentrations in TIO were normalized immediately after tumor resection, subsequently followed by normalisation of serum 1,25(OH)2D3 and phosphorus. These results suggest that FGF-23 plays an important role in the pathogenesis of hypophosphatemic rickets/osteomalacia.

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  1. National Natural Science Foundation of China,No. 30370781;Doctoral Fund of Ministry of Education of China,No. 20040023055;