Top

Published in:

01-07-2016 | Original Article

Stability and degradation of fibroblast growth factor 23 (FGF23): the effect of time and temperature and assay type

Authors: D. El-Maouche, C. E. Dumitrescu, P. Andreopoulou, R. I. Gafni, B. A. Brillante, N. Bhattacharyya, N. S. Fedarko, M. T. Collins

Published in: Osteoporosis International | Issue 7/2016

Abstract

Summary

There is growing need for a reliable assay for measuring fibroblast growth factor 23 (FGF23), a regulator of phosphorus and vitamin D. In this work, we analyze and compare the performance of three available assays, including the effect of temperature and time. This knowledge will allow for better understanding of FGF23 in the future.

Introduction

Intact and C-terminal FGF23 (iFGF23 and cFGF23) concentrations are important in the diagnosis of hypo- and hyperphosphatemic diseases. The effects of temperature, storage, and specimen handling on FGF23 levels are not well known. We investigated the effects of various factors on plasma and serum measurement of FGF23 using three different assays.

Methods

Serum and plasma FGF23 were measured using three commercially available ELISA assays—two measuring iFGF23 and one measuring cFGF23. Samples from subjects with known FGF23 disorders were stored at 4, 22, and 37 °C and analyzed at different intervals up to 48 hours (h). A subset of samples underwent repeated freeze-thaw cycles, and samples frozen at −80 °C for up to 60 months were reanalyzed. The effect of adding a furin convertase inhibitor on FGF23 degradation was investigated using samples stored at 37 °C for 48 h. Intact FGF23 levels were measured from plasma samples of four different groups to test the correlation of the two assays.

Results

Plasma FGF23 levels were stable when stored at 4 and 22 °C for 48 h. Both plasma and serum FGF23 levels demonstrated relative stability after five freeze-thaw cycles. Long-term storage at −80 °C for 40 months induced some variability in FGF23 levels. The addition of a furin inhibitor did not affect FGF23 degradation. Intact FGF23 levels showed good correlation only at the upper limit of the assay range when comparing the two assays.

Conclusions

Sample type, handling, and choice of assay are factors that affect FGF23 levels and should be considered when measuring this hormone.

(2000) Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 26:345–348

Bergwitz C, Juppner H (2009) Disorders of phosphate homeostasis and tissue mineralisation. Endocr Dev 16:133–156CrossRefPubMed

Jonsson KB, Zahradnik R, Larsson T et al (2003) Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia. N Engl J Med 348:1656–1663CrossRefPubMed

Levy-Litan V, Hershkovitz E, Avizov L et al (2010) Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am J Hum Genet 86:273–278CrossRefPubMedPubMedCentral

Riminucci M, Collins MT, Fedarko NS et al (2003) FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting. J Clin Invest 112:683–692CrossRefPubMedPubMedCentral

Benet-Pages A, Orlik P, Strom TM, Lorenz-Depiereux B (2005) An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet 14:385–390CrossRefPubMed

Lim YH, Ovejero D, Sugarman JS et al (2014) Multilineage somatic activating mutations in HRAS and NRAS cause mosaic cutaneous and skeletal lesions, elevated FGF23 and hypophosphatemia. Hum Mol Genet 23:397–407CrossRefPubMed

Shimada T, Urakawa I, Isakova T, Yamazaki Y, Epstein M, Wesseling-Perry K, Wolf M, Salusky IB, Juppner H (2010) Circulating fibroblast growth factor 23 in patients with end-stage renal disease treated by peritoneal dialysis is intact and biologically active. J Clin Endocrinol Metab 95:578–585CrossRefPubMed

Frishberg Y, Ito N, Rinat C et al (2007) Hyperostosis-hyperphosphatemia syndrome: a congenital disorder of O-glycosylation associated with augmented processing of fibroblast growth factor 23. J Bone Mineral Res: Off J Am Soc Bone Miner Res 22:235–242CrossRef

10.

Fukumoto S (2005) Post-translational modification of fibroblast growth factor 23. Ther Apher Dial: Off Peer-reviewed J Int Soc Apher Jpn Soc Apheres Jpn Soc Dial Ther 9:319–322CrossRef

11.

Kato K, Jeanneau C, Tarp MA, Benet-Pages A, Lorenz-Depiereux B, Bennett EP, Mandel U, Strom TM, Clausen H (2006) Polypeptide GalNAc-transferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O-glycosylation. J Biol Chem 281:18370–18377CrossRefPubMed

12.

Chong WH, Andreopoulou P, Chen CC et al (2013) Tumor localization and biochemical response to cure in tumor-induced osteomalacia. J Bone Miner Res: Off J Am Soc Bone Miner Res 28:1386–1398CrossRef

13.

Laboratories K FGF23 Elisa Kit. Kainos Laboratories. https://urldefense.proofpoint.com/v2/url?u=http-3A__www.kainos.co.jp_eng_products_fgf23-5Fe_fgf23-5Fe-5F5.html&d=CwIGaQ&c=y2w-uYmhgFWijp_IQN0DhA&r=DJVe1wVKJMh0si5z45T_YA&m=1G-SyHxhJ_fo6HMj693ilXk2lHraj_k71CpC9OH-9fo&s=Ru227ES1F_DD1IecUNPANWS47SCgjkC5DkAjOGRLQM8&e= 2015

14.

International I FGF23 C-terminal and Intact Immutopics. http://www.immutopics.com 2015

15.

Shrivastava A GV (2011) Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young Sci 21–25

16.

Smith ER, Ford ML, Tomlinson LA, Weaving G, Rocks BF, Rajkumar C, Holt SG (2011) Instability of fibroblast growth factor-23 (FGF-23): implications for clinical studies. Clin Chim Acta Int J Clin Chem 412:1008–1011CrossRef

17.

Smith ER, Cai MM, McMahon LP, Holt SG (2012) Biological variability of plasma intact and C-terminal FGF23 measurements. J Clin Endocrinol Metab 97:3357–3365CrossRefPubMed

18.

Jain A, McKnight DA, Fisher LW, Humphreys EB, Mangold LA, Partin AW, Fedarko NS (2009) Small integrin-binding proteins as serum markers for prostate cancer detection. Clin Cancer Res: Offi J Am Assoc Cancer Res 15:5199–5207CrossRef

19.

Heins M, Heil W, Withold W (1995) Storage of serum or whole blood samples? Effects of time and temperature on 22 serum analytes. Eur J Clin Chem Clin Biochem: J Forum Eur Clin Chem Soc 33:231–238

20.

Khosravi A, Cutler CM, Kelly MH, Chang R, Royal RE, Sherry RM, Wodajo FM, Fedarko NS, Collins MT (2007) Determination of the elimination half-life of fibroblast growth factor-23. J Clin Endocrinol Metab 92:2374–2377CrossRefPubMed

21.

Bhattacharyya N, Chong WH, Gafni RI, Collins MT (2012) Fibroblast growth factor 23: state of the field and future directions. Trends Endocrinol Metab: TEM 23:610–618CrossRefPubMedPubMedCentral

22.

Sitara D, Razzaque MS, Hesse M, Yoganathan S, Taguchi T, Erben RG, Juppner H, Lanske B (2004) Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol: J Int Soc Matrix Biol 23:421–432CrossRef

23.

Ito N, Fukumoto S, Takeuchi Y et al (2005) Comparison of two assays for fibroblast growth factor (FGF)-23. J Bone Miner Metab 23:435–440CrossRefPubMed

24.

Smith ER, McMahon LP, Holt SG (2013) Method-specific differences in plasma fibroblast growth factor 23 measurement using four commercial ELISAs. Clinical chemistry and laboratory medicine : CCLM / FESCC 51:1971–1981CrossRef

Title: Stability and degradation of fibroblast growth factor 23 (FGF23): the effect of time and temperature and assay type
Authors: D. El-Maouche
C. E. Dumitrescu
P. Andreopoulou
R. I. Gafni
B. A. Brillante
N. Bhattacharyya
N. S. Fedarko
M. T. Collins
Publication date: 01-07-2016
Publisher: Springer London
Published in: Osteoporosis International / Issue 7/2016
Print ISSN: 0937-941X
Electronic ISSN: 1433-2965
DOI: https://doi.org/10.1007/s00198-016-3543-5

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Stability and degradation of fibroblast growth factor 23 (FGF23): the effect of time and temperature and assay type

Abstract

Summary

Introduction

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Summary

Introduction

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 7/2016

The effect of two doses of dried plum on bone density and bone biomarkers in osteopenic postmenopausal women: a randomized, controlled trial

Vitamin E homologues α- and γ-tocopherol are not associated with bone turnover markers or bone mineral density in peri-menopausal and post-menopausal women

Has sclerostin a true endocrine metabolic action complementary to osteocalcin in older men?

Vertebral fracture assessment by DXA is inferior to X-ray in clinical severe osteoporosis

Hypercalcemia after discontinuation of long-term denosumab treatment

Has Choosing Wisely® affected rates of dual-energy X-ray absorptiometry use?