Benjamin D. Parker, MD; Leon J. Schurgers, PhD; Vincent M. Brandenburg, MD; Robert H. Christenson, PhD; Cees Vermeer, PhD; Markus Ketteler, MD; Michael G. Shlipak, MD, MPH; Mary A. Whooley, MD; Joachim H. Ix, MD, MAS
Fibroblast growth factor 23 (FGF23), uncarboxylated matrix Gla protein (ucMGP), and fetuin-A are regulators of mineral metabolism and inhibitors of vascular calcification. Whether circulating levels of each are associated with cardiovascular disease (CVD) events or mortality in populations without end-stage renal disease is unknown.
To evaluate the associations of FGF23, ucMGP, and fetuin-A with mortality and CVD events.
12 outpatient clinics in the San Francisco Bay area.
833 outpatients with stable coronary artery disease (CAD), recruited from 11 September 2000 to 20 December 2002.
Fibroblast growth factor 23, ucMGP, and fetuin-A concentrations were measured at baseline. Participants were followed until 1 December 2008 for mortality and CVD events.
During a median follow-up of 6.0 years, 220 participants died and 182 had CVD events. Compared with participants with FGF-23 levels in the lowest tertile, those in the highest tertile had 2-fold greater risk for mortality (hazard ratio [HR], 2.15 [95% CI, 1.43 to 3.24]) and CVD events (HR, 1.83 [CI, 1.15 to 2.91]) after adjustment for traditional CVD risk factors, C-reactive protein levels, and kidney function. The highest ucMGP tertile was associated with lower mortality risk (HR, 0.48 [CI, 0.31 to 0.75]) and showed a nonsignificant trend toward lower CVD event risk by tertile analysis (HR, 0.65 [CI, 0.40 to 1.05])â€”an association that was significant when modeled continuously (P = 0.029). No significant association of fetuin-A with mortality (HR, 0.84 [CI, 0.55 to 1.27]) or CVD events (HR, 0.99 [CI, 0.64 to 1.55]) was observed.
Participants had prevalent CAD.
In outpatients with stable CAD, higher FGF23 and lower ucMGP levels are independently associated with mortality and CVD events.
American Heart Association.
Other investigators recently identified novel regulators of mineral metabolism, which have been associated with cardiovascular disease and mortality in patients with end-stage renal disease.
The authors studied 833 persons and found that those with higher levels of fibroblast growth factor 23 and lower levels of uncarboxylated matrix Gla protein had higher cardiovascular disease event rates and mortality after adjustment for traditional risk factors, C-reactive protein levels, and kidney function.
All study participants had coronary artery disease.
Better understanding of these relationships might lead to new therapies for cardiovascular disease.
Appendix Table 1.
FGF23 = fibroblast growth factor 23; ucMGP = uncarboxylated matrix Gla protein.
* 16 participants with FGF23 levels >480 RU/mL (range, 529 to 3150 RU/mL) were not included because of space limitations.
Appendix Table 2.
Appendix Table 3.
CVD = cardiovascular disease; FGF23 = fibroblast growth factor 23; ucMGP = uncarboxylated matrix Gla protein.
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Edward R Smith
Brighton & Sussex University Hospitals NHS Trust
May 26, 2010
Important differences in Fetuin-A measurement
TO THE EDITOR:
In their recent article (1), Parker and colleagues report finding no significant association between fetuin-A levels and mortality in a cohort with prevalent coronary artery disease. Our understanding of fetuin-A biology is greatly undermined by the lack of agreement between different methods of measurement. It is particularly important therefore that method descriptions are unambiguous. The authors comment that their nephelometric fetuin-A assay "uses the same high-specificity antibody as commercially available enzyme-linked immunosorbent assays". It is unclear to which commercially available ELISAs they are referring; the two most widely-used commercial kits not only use different antisera to one another but also employ a sandwich format with two different antibodies that recognise spatially distinct molecular epitopes. This statement implies a degree of equality with other methods that we have found not to be the case (2). The authors justify the use of this method with reference to a previous study, in which use of the same fetuin-A assay demonstrated an association between fetuin-A levels and mortality in haemodialysis patients (3). However, it does not necessarily follow that this method is powered to detect significant associations in other settings, which may be masked by a lack of specificity to the biologically relevant fraction.
The poor agreement between commercial assays for fetuin-A does not represent a mere analytical nuance, but has critical implications for observations derived from their measurements. For example, in a cohort of patients with mild-to-moderate chronic kidney disease (CKD), we recently showed that fetuin-A was an independent predictor of progressive aortic stiffness (4), a vascular parameter known to be predictive of cardiovascular events and mortality. This relationship was lost, even in simple bivariate correlation, using fetuin-A measurements made with an alternative assay. Our experiments suggest that the lack of agreement in both CKD as well as non-CKD settings, reflects variation in antibody specificity for different glycosylated forms of fetuin-A (2). Although the authors of the present study rightly acknowledge that "results may differ with other assays" it remains a relatively unappreciated point that differences in antibody specificity to different modified forms of the same protein (a heterogeneous mixture) can mask potentially important associations. In the case of fetuin-A, we wonder if the investigators have considered further study using alternative methodology.
1. Parker BD, Schurgers LJ, Brandenburg VM, Christenson RH, Vermeer C, Ketteler M, et al. The associations of fibroblast growth factor 23 and uncarboxylated matrix gla protein with mortality in coronary artery disease. Ann Intern Med. 2010;152:640-8.
2. Smith ER, Ford ML, Tomlinson LA, Rocks BF, Rajkumar C, Holt SG. Poor agreement between commercial ELISAs for plasma fetuin-A: an effect of protein glycosylation? Clin Chim Acta 2010;[in press].
3. Hermans MM, Brandenburg V, Ketteler M, Kooman JP, van der Sande FM, Boeschoten EW, et al; Netherlands cooperative study on the adequacy of Dialysis (NECOSAD). Association of serum fetuin-A levels with mortality in dialysis patients. Kidney Int. 2007;72:202-7.
4. Ford ML, Tomlinson LA, Smith ER, Rajkumar C, Holt SG. Fetuin-A is an independent determinant of change of aortic stiffness over 1 year in non-diabetic patients with CKD stages 3 and Nephrol Dial Transplant. 2010;[in press].
Joachim H. Ix
University of California San Diego, and Veterans Affairs San Diego Healthcare System
August 3, 2010
In Response to Smith and Holt
Vincent M. Brandenburg, Benjamin D. Parker, and Joachim H. Ix
From the Department of Cardiology, University Hospital of the RWTH Aachen, Aachen, Germany (V.M.B), and the Department of Medicine, Division of Nephrology (B.D.P, J.H.I), and Department of Family and Preventive Medicine (J.H.I), University of California, San Diego; the Veterans Affairs San Diego Healthcare System (B.D.P, J.H.I), San Diego, California
We appreciate Drs. Smith and Holt's insightful comments about the importance of assay characteristics and we agree that associations between particular measurements and outcome might be influenced by the assay. Moreover, test specificty and reliability might depend upon factors present in the participants and specimens being evaluated.
We used a fetuin-A assay developed by our group in Aachen, Germany; an assay that is not commercially available. This assay has been described in greater detail in our prior publications(1) and was used initially to demonstrate the associations of fetuin-A with mortality in ESRD patients.(2) We evaluated the nephelometric method for AHSG serum measurement in a side-by side comparison with immunoblot analysis to exclude cross-reactivity of the antibodies with other serum proteins and proteolyitc fragments of fetuin-A. Final serum fetuin-A concentrations were calculated by regression analysis of a serial dilution curve obtained from standard serum. For comparison and reliability testing, a control solution of purified serum fetuin-A powder (Boehringer Mannheim GmbH, Mannheim, Dade-Behrging, Marburg, Germany) was prepared. For both methods, we used the polyclonal rabbit anti-human Fetuin-A antibody that does not cross-react with fetuin-B.
In regards to similarity in findings with other groups and assays, we have demonstrated that higher fetuin-A levels were associated with the metabolic syndrome,(1) and diabetes using this assay.(3) Moreover, we have demonstrated that lower fetuin-A levels were associated with aortic stenosis; an association that was present only in non-diabetic participants.(4) These data are similar to recent findings from Drs. Smith and Holt's group using the Biovendor fetuin-A assay, where lower fetuin-A levels were associated with progression of arterial stiffness (potentially a consequence of greater arterial calcium deposition); an association that was also limited to individuals without diabetes.(5) Nonetheless, we are uncertain of the correlation of our assay with that from Biovendor. We agree that future studies with multiple assays may be useful to demonstrate their correlations one to another, and to determine which may have the strongest associations with arterial calcification, arterial stiffness, and CVD events in different settings.
1. Ix JH, Shlipak MG, Brandenburg VM, Ali S, Ketteler M, Whooley MA. Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study. Circulation. Apr 11 2006;113(14):1760-1767.
2. Ketteler M, Bongartz P, Westenfeld R, et al. Association of low fetuin- A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study. Lancet. Mar 8 2003;361(9360):827- 833.
3. Parker BD, Schurgers LJ, Brandenburg VM, et al. The associations of fibroblast growth factor 23 and uncarboxylated matrix Gla protein with mortality in coronary artery disease: the Heart and Soul Study. Ann Intern Med. May 18;152(10):640-648.
4. Ix JH, Chertow GM, Shlipak MG, Brandenburg VM, Ketteler M, Whooley MA. Association of fetuin-A with mitral annular calcification and aortic stenosis among persons with coronary heart disease: data from the Heart and Soul Study. Circulation. May 15 2007;115(19):2533-2539.
5. Ford ML, Tomlinson LA, Smith ER, Rajkumar C, Holt SG. Fetuin-A is an independent determinant of change of aortic stiffness over 1 year in non- diabetic patients with CKD stages 3 and 4. Nephrol Dial Transplant. Jun;25(6):1853-1858.
Parker BD, Schurgers LJ, Brandenburg VM, Christenson RH, Vermeer C, Ketteler M, et al. The Associations of Fibroblast Growth Factor 23 and Uncarboxylated Matrix Gla Protein With Mortality in Coronary Artery Disease: The Heart and Soul Study. Ann Intern Med. ;152:640–648. doi: 10.7326/0003-4819-152-10-201005180-00004
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Published: Ann Intern Med. 2010;152(10):640-648.
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