Clinical Chemistry
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Clinical Chemistry 28: 2225-2228, 1982;
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Clinical Chemistry, Vol 28, 2225-2228, Copyright © 1982 by American Association for Clinical Chemistry

Specific spectrophotometry of ascorbic acid in serum or plasma by use of ascorbate oxidase

TZ Liu, N Chin, MD Kiser and WN Bigler

We describe a specific enzymatic spectrophotometric method for ascorbic acid in serum or plasma. Samples are analyzed indirectly by measuring the absorbance at 593 nm of a reaction product, a complex of ferrous ion and 2,4,6-tris(2-pyridyl)-s-triazine (Fe2+-TPTZ). This product is formed by reduction of the corresponding ferric ion complex (Fe3+- TPTZ), which is nonspecifically reduced by various biological reducing agents under acidic conditions. Ascorbic acid is specifically quantified by pretreating one of a pair of replicate samples with ascorbate oxidase (EC 1.10.3.3), to oxidize the ascorbic acid, then reacting both samples with Fe3+-TPTZ and measuring the difference between the absorbances at 593 nm of the treated and untreated samples. This difference is linearly related to ascorbic acid concentrations from 10 to 100 mg/L. Ten repeat determinations of a serum pool with added ascorbic acid yielded a CV of 2.8% and a mean of 47.2 mg/L. The correlation (r) between the proposed method and the dinitrophenylhydrazine method was 0.93 for 32 samples analyzed by both methods. The present method is specific for ascorbic acid and requires no deproteinization.




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Copyright © 1982 by the American Association for Clinical Chemistry.