Since none of the commercially available FGF23 assays have been validated for clinical use, FGF23 is not presently applicable for routine clinical practices. For the measurement of FGF23, four immunoassays are commercially available: Immutopics (1st and 2nd generation, San Clemente, CA, USA), Kainos (Tokyo, Japan), Millipore (Billerica, MA, USA), and DiaSorin (Saluggia, Italy). The majority of assays detect the intact 251 amino acid protein (iFGF23) by simultaneously recognizing epitopes on the N- and C-terminal domains located near the proteolytic cleavage site. Additionally, Immutopics provides an assay that quantifies both iFGF23 and the C-terminal fragment (cFGF23) using two antibodies against two C-terminal epitopes. iFGF23 is measured in picograms per milliliter (pg./mL), with a normal reference range of 11.7–48.6 pg./mL in a healthy individual, whereas cFGF23 is reported in relative units (RU) per milliliter, with a normal reference range of 21.6–91.0 RU/mL
[15][47]. Due to the possibility that iFGF23 may be degraded by protease enzyme or changed after venipuncture, two iFGF23 stability studies discovered decreasing FGF23 levels following an 8-h delay in centrifugation, but no evidence of deterioration after storing processed samples at −80 °C
[16][48]. Biological variability studies in healthy individuals revealed that iFGF23 levels have a diurnal variation that peaks in the early morning and gradually declines during the day
[15][47]. In comparison, the concentrations of cFGF23 could be slightly increased throughout the day
[17][49] and see no significant change after dietary or phosphate intake
[18][50]. Despite the stability and biological variability advantages of cFGF23, cFGF23 assays may be more applicable than iFGF23 assays, particularly for diagnostic and prognostic studies. In contrast, iFGF23 may outperform in representing the biological effects of FGF23 in etiognostic and therapeutic research because the c-terminal fragments might have counter-regulatory effects on the physiologically active FGF23
[19][51]. The schematic summary of FGF23 production and its measurement are illustrated in
Figure 14.
Figure 14. FGF23 production and immunoassay measurements. (
a) After completed transcription and translation, FGF23 can be transferred to two post-translation modification pathways, including O-glycosylation with GALNT3 on Thr178, or phosphorylation by the extracellular serine/threonine protein kinase FAM20C at Ser180. O-glycosylation modification by GALANT3, stabilized form, can prevent intact FGF23 from cleavage. In contrast, phosphorylated FGF23 by FAM20C can be cleaved into N-terminal and C-terminal fragments within the osteocyte/osteoblast. These peptides, including full-length (intact) FGF23, N-terminal fragments, and C-terminal fragments, can be detected in the circulation. (
b) For C-terminal assays, detecting antibodies bind to C-terminus epitopes to detect both full-length FGF23 and its C-terminal fragments, whereas assays for intact FGF23 use antibodies to detect epitopes surrounding the FGF23 cleavage site for the detection of only full-length FGF23. This figure was generated with publication licensed by BioRender, Toronto, ON, Canada (Agreement number: DV237SONHF, 19 November 2021). Abbreviations: GALNT3, polypeptide N-acetyl galactosaminyltransferase 3; FAM20C, the extracellular protein kinase FAM20C; Ser, Serine; Thr, Threonine.
5. Conclusions
A pivotal role of FGF23 was found in local and systemic bone remodelling with supraphysiological levels causing abnormal bone formation, although any direct effect on osteoblasts remains unclear as well as a controversial links between FGF23 with osteoclastogenesis and bone resorption. Current evidence from clinical studies indicates that FGF23 could be a risk factor of bone fragility in CKD-MBD, but not a major contributor to age-related osteoporosis. An increased FGF23 level may represent an abnormal state of bone mineral homeostasis, but is not a direct indicator of decreased bone mineral density (BMD). Since clinical studies, both in healthy elderly and in patients with impaired renal function, showed that elevated FGF23 levels were an independent risk factor of fragility fracture, a future predictive model for fragility fracture may incorporate FGF23 as a factor to represent bone mineral homeostasis status. FGF23 is putative factor in the fragility of CKD-MBD, less so in age-related bone loss; future elucidation of pathogenesis requires remodelling biomarkers, while gender differences need elucidation with respect to abnormal bone mineral homeostasis and reduced BMD as part of a future model of fragility risk factors.