Instability of fibroblast growth factor-23 (FGF-23): Implications for clinical studies

doi:10.1016/j.cca.2011.02.009

Clinica Chimica Acta

Volume 412, Issues 11–12, 12 May 2011, Pages 1008-1011

https://doi.org/10.1016/j.cca.2011.02.009 Get rights and content

Abstract

Background

Fibroblast growth factor-23 (FGF-23) is a bone secreted hormone that regulates phosphate homeostasis and calcitriol levels. FGF-23 concentrations are elevated in chronic kidney disease (CKD), oncogenic osteomalcia and a number of rare hereditary disorders. Studies systematically evaluating the pre-analytical stability of intact FGF-23 are lacking.

Methods

The stability of FGF-23 was assessed in timed experiments using blood taken into K₂-EDTA plasma specimen tubes from a group of healthy participants and from a group with mild-to-moderate CKD. We evaluated the use of aprotinin, a serine protease inhibitor, and a commercially available protease inhibitor cocktail to preserve intact FGF-23 after blood collection. FGF-23 measurements were made using both intact and C-terminal assays.

Results

Both whole blood and separated sample studies demonstrated a rapid loss of intact FGF-23 within 2 h, while concentrations increased using the C-terminal assay. The addition of protease inhibitor cocktail stabilised FGF-23 concentrations for 4 h after blood collection. Intact and C-terminal assay FGF-23 measurements showed poor correlation in both healthy and CKD cohorts.

Conclusion

K₂-EDTA plasma samples, even when promptly separated, are unsuitable for measurement of FGF-23 unless stabilised with a protease inhibitor cocktail.

Introduction

Fibroblast growth factor-23 (FGF-23) is a bone secreted hormone that regulates urinary phosphate excretion and calcitriol levels [1]. FGF-23 concentrations are elevated in oncogenic osteomalcia (or tumour-induced osteomalacia) and a number of rare hereditary disorders [2].

FGF-23 levels increase markedly with failing renal function and are frequently 100- to 10,000 fold above that of healthy controls in end-stage renal disease (ESRD) [3], [4]. In this setting, circulating FGF-23 concentrations have been independently associated with disease progression [5], vascular dysfunction [6], left ventricular hypertrophy [7] and mortality [8].

In vitro studies show that full-length (Y25 to I251) or ‘intact’ FGF-23 activity is partly regulated through post-synthetic proteolytic cleavage at the furin proconvertase recognition site ¹⁷⁶RXXR¹⁷⁹ [9]. It is not currently clear whether the N- and C-terminal fragments generated by proteolysis are themselves biologically active. Two-site (sandwich) ELISA kits are commercially available that detect only intact FGF-23 or that recognize both intact and C-terminal fragments (‘C-terminal’ assay). Although there are reports showing highly significant correlations between FGF-23 measurements made using intact and C-terminal assays in CKD patients undergoing dialysis [8], [10], [11] it is not known whether measurements are in such close agreement in other settings.

We sought to assess the short-term stability of FGF-23 in whole blood and plasma, and to investigate the use of protease inhibitors to reduce loss of intact protein. Further, having demonstrated a method of stabilising intact FGF-23 levels, we sought to assess the agreement of intact and C-terminal concentrations in patients with mild-to-moderate CKD and in a group of healthy participants.

Section snippets

Study participants

We recruited 40 healthy non-smoking, non-diabetic individuals (mean age 45 ± 8 years, 18 male) with normal renal function (4-variable Modification of Diet in Renal Disease (MDRD) derived eGFR > 60 mL/min/1.73 m², uPCR < 30 mg/mmol) and no history of cardiovascular disease (angina, previous myocardial infarction, stroke or heart failure, hypertension or dyslipidaemia requiring therapy). For each healthy volunteer, we collected venous blood 4 mL K₂-EDTA specimen tubes (Griener Vacuette System, UK). After

FGF-23 stability in whole blood and separated plasma

In whole blood stability studies (n = 15), trend analysis of the change in intact FGF-23 concentration showed a significant decrease in K₂-EDTA plasma samples over 4 h (P < 0.001, Fig. 1A). Likewise, in promptly separated K₂-EDTA plasma samples, intact FGF-23 concentrations significantly decreased over the same time period (P < 0.001). Of note, the magnitude of the apparent loss of intact FGF-23 immunoreactivity in K₂-EDTA plasma was not significantly different to the decrease seen in whole blood

Discussion

This is the first study to systematically evaluate the short-term stability of FGF-23 post venesection in healthy and pre-dialysis CKD groups. We have demonstrated that there is a marked loss of intact FGF-23 within 2 h of blood-taking. Conversely, we found a significant increase in FGF-23 concentrations when measured using the C-terminal assay over the same time course.

Given that the C-terminal assay detects both intact and C-terminal fragments, one might expect this assay signal to remain

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Pre-analytical stability of FGF23 with the contemporary immunoassays
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For clarity and practicality, we here distinguish immediate pre-centrifugation stability (centrifugation <1 h), delayed centrifugation stability (centrifugation after 8 h) and delayed storage stability (after centrifugation) of FGF23. In 2011, Smith et al. first assessed the stability of FGF23 at room temperature both in whole blood and in promptly centrifuged plasma using the two 1st generation assays from Immutopics (the intact and C-terminal FGF23 assays) [6]. They reported higher baseline concentrations of intact FGF23 at 30 min when collection tubes were pre-coated with a protease inhibitor cocktail or aprotinin, suggestive of immediate protein proteolysis in tubes that did not contain protease inhibitors.
Several publications have reported on the pre-analytical stability of fibroblast growth factor 23 (FGF23) and some recommend coating blood collecting tubes with protease inhibitors, in order to prevent degradation. These recommendations are based on observations for a first generation assay for the measurement of intact FGF23. However, if this also applies for the contemporary immunoassays, and at what stage of pre-analysis, is unknown.
We reviewed data from these previous reports on the issue of FGF23 stability and complemented these findings with data from novel experiments.
We concluded that the contemporary intact FGF23 assays by Immutopics, Kainos, Millipore and DiaSorin do not suffer from immediate loss of FGF23 signal and do not require blood withdrawal in protease inhibitor-coated collecting tubes. Nevertheless, FGF23 concentrations do decline when centrifugation is delayed up to 8 h and prompt centrifugation is therefore advised.
FGF23: Clinical usefulness and analytical evolution
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Accordingly, this later kit does not allow to differentiate the relative levels of iFGF23 and cFGF23. Analytical agreement between these two assays seems poor, particularly in healthy subjects [21,24,35,76,77]. It could be explained by a lower stability of the intact form after blood collection in a tube without protease inhibitor.
Fibroblast Growth Factor 23 (FGF23) is a key hormone for the regulation of phosphate homeostasis. Over the past decades, FGF23 was the subject of intense research in the fields of nephrology and the cardiology. It presents a remarkable correlation with well-established biomarkers of cardiovascular disorders in both chronic kidney disease (CKD) and heart failure (HF) patients. The interest of FGF23 lies in its early-onset in the primary course of CKD as well as in the incremental prognosis information it conveys in both CKD and HF.
Different types of assays of FGF-23 testing exist, those targeting the intact form (iFGF23), the other one detecting terminal fragments (cFGF23). The issue is still pending which assay suits best for clinical use. Recently, the implementation of this biomarker on multianalyzer platforms, on which other markers of phospho-calcic balance are set up, allows a rapid turn-around-time and a potential financial gain. However, despite the good analytical performances of the automated methods, there is a poor harmonization between assays. The introduction of an international certified reference material should standardize the measurement and improve the harmonization of results from different laboratories.
A deeper understanding of physio-pathological mechanisms and processing of FGF-23 should reinforce its clinical indications and might also identify new therapeutic targets for the treatment of CKD and HF.
Clinical utility of bone markers in various diseases
2018, Bone
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Regarding the FGF23, both intact and C-terminal FGF23 assays can be used to measure FGF23. The C-terminal assay, which detects both intact FGF23 and its C-terminal fragment, is often the assay of choice due to less significant diurnal pattern and also less pre-analytical precautions [97]. However, the intact FGF23 is thought to be the biologically active form [42, 53, 54].
Measurements of bone markers (BMs) in peripheral blood or urine are a pivotal part of bone research within modern clinical medicine. In recent years the use of BMs increased substantially as they can be useful either to diagnose bone (related) disease and to follow its natural history, but also to monitor the effects of interventions. However, the use of BMs is still complicated mainly due to (pre)analytical variability of these substances, limited accessibility of assays, variable cut-off values in different countries and laboratories and heterogeneous results with regard to clinical implications of measuring BMs in several studies. This review will provide the clinician with a practical guide, based on current evidence, in which circumstances to test which bone markers for optimal diagnostic purposes, in order to improve patient care in different areas of bone diseases including Paget's disease, primary osteoporosis, tumor induced osteomalacia, hypophosphatemic rickets, van Buchem disease, chronic kidney disease, rheumatoid arthritis, neoplasma/multiple myeloma, type 2 diabetes mellitus and primary hyperparathyroidism. The clinician should consider fasting state, recent fractures, aging, menopausal status, concomitant liver and kidney disease when ordering and interpreting BM measurements as these factors might result in misleading BM concentrations. We found that BMs are clearly useful in the current diagnosis of tumor induced osteomalacia, van Buchem disease, Paget's disease and hypophosphatemic rickets. In addition, BMs are useful to monitor disease activity in chronic kidney disease, Paget's disease and are useful to monitor treatment adherence in osteoporosis.
Fibroblast growth factor 23 and 25-hydroxyvitamin D levels are associated with estimated glomerular filtration rate decline
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We used this assay, because C-terminal FGF23 fragments were reported to be present as circulating FGF23 in serum from patients with end-stage renal disease.30 Moreover, unlike in hemodialysis patients, the correlation between the two assays is poor in pre-dialysis patients.3132 Serum Ca levels were corrected for Alb using the following formula (corrected Ca=total Ca+(4.0-Alb) × 0.8, if Alb<4.0 g/dl).
The combination of serum 25-hydroxyvitamin D (25D) and fibroblast growth factor 23 (FGF23) levels predict hard renal outcomes in patients with chronic kidney disease (CKD), independent of classical markers of mineral and bone disorders, including serum phosphorus, parathyroid hormone, 1,25-dihydroxyvitamin D levels, and active vitamin D therapy. In a prospective cohort study of 738 Japanese pre-dialysis outpatients with CKD, we examined potentially non-linear associations between 25D and FGF23 levels and estimated glomerular filtration rate (eGFR) changes in 727 patients with at least a 6-month observation period and no history of admission by acute kidney injury. We used multiple regression analyses with restricted cubic spline functions using annualized eGFR decline as a dependent variable. A significantly non-linear positive relationship between 25D and eGFR changes was observed. The annualized eGFR decline was greater in patients with 25D concentrations <25 and 23 ng/ml in univariate and multivariate analyses, respectively. Above this threshold, the eGFR decline plateaued. FGF23 showed a linear negative association with eGFR changes. After dividing the patients into four groups according to median 25D and FGF23 levels, the annualized eGFR changes in the Low FGF23-Low 25D, High FGF23-High 25D, and High FGF23-Low 25D groups were 0.49 (95% confidence intervals: -2.83 to 3.81), -1.24 (-5.00 to 2.52), -4.77 (-8.85 to -0.69), respectively, relative to the Low FGF23-High 25D group (P for trend, 0.02). Thus, combined use of FGF23 and 25D is useful to predict eGFR change in patients with CKD as well as hard renal outcomes.
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Instability of fibroblast growth factor-23 (FGF-23): Implications for clinical studies

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Study participants

FGF-23 stability in whole blood and separated plasma

Discussion

Kidney Int

Kidney Int

Bone

Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism

J Clin Invest

Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia

N Engl J Med

Fibroblast growth factor-23 mitigates hyperphosphatemia but accentuates calcitriol deficiency in chronic kidney disease

J Am Soc Nephrol

Fibroblast growth factor 23 (FGF23) predicts progression of chronic kidney disease: the Mild to Moderate Kidney Disease (MMKD) Study

J Am Soc Nephrol