The Effect of Anticoagulants on Zinc Measurement: A Comparative Study in Serum and Plasma Samples Using a Colorimetric Method
Abstract
Objectives: Zinc (Zn) is a vital trace element, and its accurate measurement in biological samples is important for diagnosing Zn deficiency. However, the difference in Zn concentration between serum and plasma samples and the effect of anticoagulants on results are major methodological challenges. The aim of this study was to compare the effect of common anticoagulants (EDTA, citrate and heparin) on Zn concentration in blood samples using a routine colorimetric method.
Methods: This research was conducted on 20 healthy volunteers (10 women and 10 men). Samples of serum, EDTA, citrate and heparin plasma were collected and Zn levels were measured using a routine colorimetric method based on 5‑Br‑PAPS.
Results: The results of this study showed that Zn concentrations in EDTA‑ and citrate‑anticoagulated plasma were significantly lower than those measured in serum (P<0.05). In contrast, no statistically significant difference was observed between serum and heparin‑anticoagulated plasma (P>0.05). Spearman’s correlation analysis showed a strong positive correlation between serum Zn concentrations and those measured in heparin (r=0.831, P<0.001) and citrate (r=0.704, P<0.01) plasma samples, whereas the correlation between serum and EDTA plasma was positive but weak and not statistically significant (r=0.099, P>0.05).
Conclusion: The choice of anticoagulant is a critical pre-analytical factor in Zn measurement using colorimetric methods. These findings show that chelating anticoagulants like EDTA and citrate spuriously lower Zn concentration, whereas heparin‑anticoagulated plasma yields Zn measurements that are comparable to those obtained in serum. These results are of great importance for the accurate interpretation of results in clinical laboratories.
2. Prasad AS. Zinc in human health: effect of zinc on immune cells. Mol Med. 2008;14(5-6):353-7. https://doi.org/10.2119/2008-00033.Prasad.
3. Killilea DW, Schultz K. Pre-analytical variables influence zinc measurement in blood samples. PLoS One. 2023;18(9):e0286073. https://doi.org/10.1371/journal.pone.0286073.
4. Smith JC, Holbrook JT, Danford DE. Analysis and evaluation of zinc and copper in human plasma and serum. J Am Coll Nutr. 1985;4(6):627-38. https://doi.org/10.1080/07315724.1985.10720105.
5. Keyzer J, Oosting E, Wolthers B, Muskiet F, Hindriks F, Van der Slik W. Zinc in plasma and serum: influence of contamination due to the collection tubes. Pharmaceutisch Weekblad. 1983;5(5):248-51.
6. Chen WJ, Zhao CY, Zheng TL. Comparison of zinc contents in human serum and plasma. Clin Chim Acta. 1986;155(2):185-7. https://doi.org/10.1016/0009-8981(86)90282-2.
7. Nyborg JK, Peersen OB. That zincing feeling: the effects of EDTA on the behaviour of zinc-binding transcriptional regulators. Biochem J. 2004;381(3):e3. https://doi.org/10.1042/bj20041096
8. Gervin CA, Gervin AS, Nichols W, Corrigan Jr JJ. Problems in the measurement of zinc using heparin as an anticoagulant. Life Sci. 1983;33(26):2643-9. https://doi.org/10.1016/0024-3205(83)90348-x
9. Frank EL, Hughes MP, Bankson DD, Roberts WL. Effects of anticoagulants and contemporary blood collection containers on aluminum, copper, and zinc results. Clin Chem. 2001;47(6):1109-12. https://doi.org/10.1093/clinchem/47.6.1109
10. Jablan J, Grdić Rajković M, Inić S, Petlevski R, Domijan A-M. Impact of anticoagulants on assessment of zinc in plasma. Croatica Chem Acta. 2018;91(3):317-21. https://doi.org/10.5562/cca3321
11. Fuwa K, Pulido P, McKay R, Vallee BL. Determination of Zinc in Biological Materials by Atomic Absorption Spectrometry. Anal Chem. 1964;36(13):2407-11. https://doi.org/10.1021/ac60219a009
12. Laur N, Kinscherf R, Pomytkin K, Kaiser L, Knes O, Deigner HP. ICP-MS trace element analysis in serum and whole blood. PLoS One. 2020;15(5):e0233357. https://doi.org/10.1371/journal.pone.0233357.
13. Prasad AS. Impact of the discovery of human zinc deficiency on health. J Am Coll Nutr. 2009;28(3):257-65. https://doi.org/10.1080/07315724.2009.10719780.
14. Wieringa FT, Dijkhuizen MA, Fiorentino M, Laillou A, Berger J. Determination of zinc status in humans: which indicator should we use? Nutrients. 2015;7(5):3252-63. https://doi.org/10.3390/nu7053252
15. King JC, Brown KH, Gibson RS, Krebs NF, Lowe NM, Siekmann JH, Raiten DJ. Biomarkers of Nutrition for Development (BOND)-Zinc Review. J Nutr. 2015;146(4):858S-85S. https://doi.org/10.3945/jn.115.220079.
16. Gordon SJV, Xiao Y, Paskavitz AL, Navarro-Tito N, Navea JG, Padilla-Benavides T. Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells. J Vis Exp. 2019(147):e59519. https://doi.org/10.3791/59519.
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| Issue | Vol 3 No 4 (2025) | |
| Section | Original Articles | |
| Keywords | ||
| Zinc Anticoagulant Colorimetric method Serum Plasma | ||
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