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Intact fibroblast growth factor 23 and fragments in plasma from Gambian children
V Braithwaite1, S F A Bruggraber, A Prentice
1MRC Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge CB1 9NL, United Kingdom. vickie.braithwaite@mrc-hnr.cam.ac.uk
Insights
Elevated Fibroblast Growth Factor 23 (FGF23) in Gambian children with rickets is due to intact FGF23 hormone, not inactive fragments. Western blotting confirmed only intact FGF23 was present in plasma samples.
Area of Science:
- Biochemistry
- Pediatric Endocrinology
- Nutritional Science
Background:
- Elevated Fibroblast Growth Factor 23 (FGF23) concentrations are observed in Gambian children, with higher levels in those with rickets.
- The C-terminal Immutopics enzyme-linked immunosorbent assay (ELISA) detects both intact FGF23 and C-terminal fragments, necessitating further investigation into the composition of circulating FGF23.
Purpose of the Study:
- To investigate whether elevated FGF23 levels in Gambian children are primarily due to intact FGF23 hormone or C-terminal FGF23 fragments.
- To validate the findings of the C-terminal Immutopics ELISA using western blotting.
Main Methods:
- Western blotting was employed to analyze stored plasma samples from Gambian children with and without rickets-like bone deformities.
- Samples were selected based on FGF23 concentrations determined by C-terminal Immutopics ELISA (elevated >900 RU/ml, normal <30 RU/ml).
- An anti-FGF23 polyclonal antibody recognizing the C-terminal was used as the primary antibody.
Main Results:
- C-terminal FGF23 fragments, detectable in ELISA standards, were absent in the plasma samples from Gambian children.
- Western blotting revealed no discernible difference in the size of FGF23 molecules between children with and without rickets-like bone deformities.
- Only intact FGF23 hormone was detected in all analyzed plasma samples.
Conclusions:
- The elevated FGF23 concentrations measured by the C-terminal Immutopics ELISA in Gambian children are attributable to intact FGF23 hormone.
- The study provides evidence that increased circulating inactive C-terminal fragments do not account for the elevated FGF23 levels observed in this population.
- Western blotting confirms the presence of intact FGF23 as the predominant form in Gambian children's plasma, regardless of rickets status.
Unlabelled:
Fibroblast growth factor 23 (FGF23) is grossly elevated in Gambian children with rickets and, at a lower prevalence, in those without bone deformities. We used western blotting to mimic the detection capabilities of the C-terminal FGF23 enzyme-linked immunosorbent assay (ELISA). Only intact FGF23 hormone was present in Gambian plasma samples from children with and without rickets.
Introduction:
Elevated circulating FGF23 concentrations have been detected in plasma samples from Gambian children using the C-terminal Immutopics ELISA. The Immutopics ELISA detects both the intact FGF23 hormone and the C-terminal fragment. The aim of this study was to determine whether the elevated FGF23 concentrations as detected by the ELISA were predominantly due to a high proportion of intact FGF23 hormone and/or C-terminal FGF23 fragments.
Methods:
Stored, frozen plasma samples from previous studies of Gambian children with known concentrations of FGF23 as determined by C-terminal Immutopics ELISA assay, were selected for western blotting analysis: from children with rickets-like bone deformities (n = 4) and local controls (n = 4), with elevated >900 RU/ml (n = 2) and normal <30 RU/ml (n = 2; from each group). The anti-FGF23 polyclonal antibody that recognizes the C-terminal of FGF23 (as used in the Immutopics kit) was used as the primary antibody and the anti-IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) was used as the secondary antibody.
Results:
Firstly, C-terminal FGF23 fragments, although detectable in standards from the Immutopics ELISA kit, were not in the Gambian plasma samples. Secondly, there was no difference in the size of FGF23 molecules present in plasma from children with rickets-like bone deformities and children from the local community.
Conclusions:
Western blotting has provided evidence that elevated FGF23 concentrations, as determined by the C-terminal Immutopics ELISA, measured in Gambian children with and without rickets-like bone deformities was not caused by an increased proportion of circulating inactive C-terminal fragments.
