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False in vitro and in vivo elevations of uric acid levels in mouse blood
Tamaki Watanabe1, Naoko H Tomioka, Shigekazu Watanabe
1a Faculty of Pharma-Sciences, Practical Pharmacy , Teikyo University , Tokyo , Japan.
Abstract:
Uric acid (UA) levels in mouse blood have been reported to range widely from 0.1 μM to 760 μM. The aim of this study was to demonstrate false in vitro and in vivo elevations of UA levels in mouse blood. Male ICR mice were anesthetized with pentobarbital (breathing mice) or sacrificed with overdose ether (non-breathing mice). Collected blood was dispensed into MiniCollect® tubes and incubated in vitro for 0 or 30 min at room temperature. After separation of plasma or serum, the levels of UA and hypoxanthine were determined using HPLC. From the non-incubated plasma of breathing mice, the true value of UA level in vivo was 13.5±1.4 μM. However, UA levels in mouse blood increased by a factor of 3.9 following incubation in vitro. This "false in vitro elevation" of UA levels in mouse blood after blood sampling was inhibited by allopurinol, a xanthine oxidase inhibitor. Xanthine oxidase was converted to UA in mouse serum from hypoxanthine which was released from blood cells during incubation. Plasma UA levels from non-breathing mice were 19 times higher than those from breathing mice. This "false in vivo elevation" of UA levels before blood sampling was inhibited by pre-treatment with phentolamine, an α-antagonist. Over-anesthesia with ether might induce α-vasoconstriction and ischemia and thus degrade intracellular ATP to UA. For the accurate measurement of UA levels in mouse blood, the false in vitro and in vivo elevations of UA level must be avoided by immediate separation of plasma after blood sampling from anesthetized breathing mice.
Insights
Accurate uric acid (UA) measurement in mice requires avoiding false elevations. This study reveals in vitro and in vivo methods that inflate UA levels, necessitating immediate plasma separation from anesthetized, breathing mice.
Area of Science:
- Biochemistry
- Animal Physiology
- Analytical Chemistry
Background:
- Uric acid (UA) levels in mouse blood exhibit wide variability, complicating accurate measurement.
- Previous studies have not fully addressed the causes of artificially elevated UA levels in mouse blood.
- Understanding factors influencing UA levels is crucial for reliable experimental outcomes.
Purpose of the Study:
- To investigate and demonstrate false in vitro and in vivo elevations of uric acid (UA) levels in mouse blood.
- To identify methods to prevent these artificial increases in UA measurements.
- To establish accurate protocols for UA quantification in mouse blood samples.
Main Methods:
- Male ICR mice were anesthetized with pentobarbital or sacrificed with ether.
- Blood samples were collected and either immediately processed or incubated in vitro.
- Uric acid and hypoxanthine levels were quantified using High-Performance Liquid Chromatography (HPLC).
Main Results:
- In vitro incubation of mouse blood led to a 3.9-fold increase in UA levels, inhibited by allopurinol.
- Xanthine oxidase converted hypoxanthine released from blood cells into UA during incubation.
- Plasma UA levels in non-breathing mice were 19 times higher than in breathing mice, an effect inhibited by phentolamine.
Conclusions:
- False in vitro elevations of UA result from hypoxanthine conversion in serum during incubation.
- False in vivo elevations are linked to ether anesthesia-induced vasoconstriction and ATP degradation to UA.
- Accurate mouse blood UA measurement requires immediate plasma separation from anesthetized, breathing mice.
