TY - JOUR
T1 - Antioxidant properties of citric acid interfere with the uricase-based measurement of circulating uric acid
AU - Ryan, Evan M.
AU - Duryee, Michael J.
AU - Hollins, Andrew
AU - Dover, Susan K.
AU - Pirruccello, Samuel Jay
AU - Sayles, Harlan
AU - Real, Kevin D.
AU - Hunter, Carlos D.
AU - Thiele, Geoffrey Milton
AU - Mikuls, Ted R
N1 - Funding Information:
This research was supported by the University of Nebraska Medical Center Enhanced Medical Education Tract in Autoimmune Diseases . Dr. Mikuls is supported by grants from the National Institute of General Medical Sciences ( U54GM115458 ); National Institute on Alcohol Abuse and Alcoholism ( R25AA020818 ) and National Institute of Arthritis and Musculoskeletal and Skin Diseases ( 2P50AR60772 ).
Publisher Copyright:
© 2018 Elsevier B.V.
PY - 2019/2/5
Y1 - 2019/2/5
N2 - 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.
AB - 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.
KW - Antioxidant
KW - Citric acid
KW - Hydrogen peroxide
KW - Sodium citrate
KW - Uric acid measurement
KW - Uricase method
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U2 - 10.1016/j.jpba.2018.11.011
DO - 10.1016/j.jpba.2018.11.011
M3 - Article
C2 - 30447534
AN - SCOPUS:85056600459
SN - 0731-7085
VL - 164
SP - 460
EP - 466
JO - Journal of Pharmaceutical and Biomedical Analysis
JF - Journal of Pharmaceutical and Biomedical Analysis
ER -