Isolation and identification of alpha-CEHC sulfate in rat urine and an improved method for the determination of conjugated alpha-CEHC.
2,5,7,8-Tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the water-soluble metabolite of alpha-tocopherol (alpha-TOH) with a shortened side chain but an intact hydroxychroman structure, has been identified in human urine and are thought to be produced in significant amount at excess intake of alpha-TOH. In previous studies, CEHCs in biological specimens were measured by HPLC, GC-MS or LC-MS, preceded by a hydrolysis procedure using either enzyme or methanolic HCl. In an attempt to analyze alpha-CEHC in rat urine accordingly, we observed that enzyme hydrolysis was relatively inefficient in releasing alpha-CEHC compared to high concentrations of HCl. The HCl releasable alpha-CEHC conjugate was isolated and chemically identified as 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate). Using the synthetic alpha-CEHC sulfate standard, it was found that sulfatase could not hydrolyze to a significant extent. On the other hand, pretreatment with HCl at 60 degrees C in the presence of ascorbate, followed by a one-step ether extraction, not only hydrolyzed the sulfate conjugate completely but also extracted alpha-CEHC with high recovery. The inclusion of ascorbate minimized the conversion of alpha-CEHC to alpha-tocopheronolactone in the HCl pretreatment. A complete procedure for the quantitative analysis of alpha-CEHC including HCl hydrolysis, ether extraction and reverse phase isocratic HPLC-ECD was thus established. In conclusion, alpha-CEHC sulfate was isolated and identified as the HCl-releasable conjugate of alpha-CEHC in rat urine. A rapid and sensitive method with high reproducibility for the determination of free, conjugated and total alpha-CEHC is then established.
Li
Yi-Jen
YJ
Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan.
Luo
Sheng-Ching
SC
Lee
Yi-Jing
YJ
Lin
Fu-Jung
FJ
Cheng
Chi-Cheng
CC
Wein
Yung-Shung
YS
Kuo
Yueh-Hsiung
YH
Huang
Ching-jang
CJ
D000818:Animals; D002839:Chromans / Q000737:chemistry* / Q000302:isolation & purification* / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006851:Hydrochloric Acid / Q000737:chemistry; D006868:Hydrolysis; D011422:Propionates / Q000737:chemistry* / Q000302:isolation & purification* / Q000652:urine; D051381:Rats; D017208:Rats, Wistar; D024502:alpha-Tocopherol / Q000378:metabolism
D002839:Chromans; D011422:Propionates; C084852:2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman; D024502:alpha-Tocopherol; D006851:Hydrochloric Acid