Antioxidant properties of citric acid interfere with the uricase-based measurement of circulating uric acid

Evan M. Ryan, Michael J. Duryee, Andrew Hollins, Susan K. Dover, Samuel Jay Pirruccello, Harlan Sayles, Kevin D. Real, Carlos D. Hunter, Geoffrey Milton Thiele, Ted R Mikuls

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Background: Circulating uric acid (UA) is an important biomarker, not only in the detection and management of gout, but also in assessing the risk of related comorbidity. The impact of collection methods on clinical UA measurements has been the subject of limited study. After observing significant differences between UA concentrations of blood samples obtained by different collection tubes, we began examining the effects of exogenous tube components on measured UA concentrations. We aimed to: (1) demonstrate the variability in uricase-based UA measurements attributable to different collection methods and (2) identify factors influencing this variability. Methods: Blood samples from human subjects were collected using Serum Separator Tubes (SST tubes), Acid Citrate Dextrose (ACD) tubes, and Sodium Citrate (SC) tubes. Circulating UA concentrations were measured by chemistry analyzers utilizing the uricase method. Absorbance assays were run in order to determine the effects of citric acid, sodium citrate, and dextrose on measured absorbance in the presence of leuco crystal violet dye, hydrogen peroxide, and peroxidase. Statistical analyses—including Student's T tests and ANOVA—were used to compare results. Results: UA concentrations of blood samples collected in ACD tubes were significantly lower than those collected in SST tubes (P < 0.01). Samples collected in SC tubes trended towards lower UA measurements than samples collected in SST tubes, although this difference did not reach statistical significance (P = 0.06). Blood samples spiked with separate concentrations of sodium citrate (3.2 and 22.0 g/L), citric acid (8.0 g/L), and dextrose (24.5 g/L) demonstrated significantly lower UA measurements compared to controls (P < 0.01). Absorbance assays demonstrated that increasing concentrations of citric acid and sodium citrate—in the presence of leuco crystal violet, hydrogen peroxide, and peroxidase—decreased the amount of oxidized dye in the uricase method of UA measurement in a dose-dependent manner (P < 0.01). In contrast, dextrose did not significantly alter the amount of oxidized dye available. Discussion: Our results indicate that citric acid obstructs accurate uricase-based UA measurement, providing falsely low values. Citric acid, a known antioxidant, scavenges hydrogen peroxide, a key intermediate using the uricase method. By scavenging hydrogen peroxide, citric acid decreases the amount of oxidized leuco dye leading to falsely low UA measurements. Therefore, collection tubes, like ACD and SC tubes, which contain concentrations of citric acid or its conjugate base sodium citrate should not be used to measure circulating UA levels when utilizing uricase-based measurement methods.

Original languageEnglish (US)
Pages (from-to)460-466
Number of pages7
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume164
DOIs
StatePublished - Feb 5 2019

Keywords

  • Antioxidant
  • Citric acid
  • Hydrogen peroxide
  • Sodium citrate
  • Uric acid measurement
  • Uricase method

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

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