Determination of dehydroascorbic acid in mouse tissues and plasma by using tris(2-carboxyethyl)phosphine hydrochloride as reductant in metaphosphoric acid/ethylenediaminetetraacetic acid solution.
Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals in vitro. Moreover, AA is an essential ingredient for post-translational proline hydroxylation of collagen molecules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we describe an improved method for assessing DHA in biological samples. The use of 35 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution of metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method enabled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebellum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this improved method provides a useful standard for all DHA determinations.
Sato
Yasunori
Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, Chiba 274-8510, Japan.
Uchiki
Takayuki
T
Iwama
Mizuki
M
Kishimoto
Yuki
Y
Takahashi
Ryoya
R
Ishigami
Akihito
A
D000818:Animals; D000975:Antioxidants / Q000032:analysis* / Q000378:metabolism; D001205:Ascorbic Acid / Q000032:analysis* / Q000378:metabolism; D019411:Clinical Laboratory Techniques / Q000379:methods*; D003683:Dehydroascorbic Acid / Q000032:analysis* / Q000097:blood; D004492:Edetic Acid / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009928:Organ Specificity; D018384:Oxidative Stress; D010720:Phosphines / Q000032:analysis*; D010757:Phosphorous Acids / Q000494:pharmacology; D019163:Reducing Agents / Q000494:pharmacology
D000975:Antioxidants; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid; D004492:Edetic Acid; D010720:Phosphines; D010757:Phosphorous Acids; D019163:Reducing Agents; C080938:tris(2-carboxyethyl)phosphine; C043639:metaphosphoric acid