| Project/Area Number |
61870018
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | University of Tokyo |
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Principal Investigator |
SEYAMA Yousuke Univ. Tokyo, Faculty of Med., Professor, 医学部, 教授 (90010082)
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| Co-Investigator(Kenkyū-buntansha) |
楠 慎一郎 アドバンス株式会社, 生命科学研究所, 所長
OTSUKA Hideaki Hiroshima Univ. School of Med., Assoc, Prof., 医学部, 助教授 (00107385)
KAMEI Sachiko Univ. Tokyo, Faculty of Med., Lecturer, 医学部, 講師 (40107503)
KASAMA Takeshi Univ. Tokyo, Faculty of Med., Associate, 医学部, 教務職 (80124668)
SHIMIZU Takao Univ. Tokyo, Faculty of Med., Assoc. Prof., 医学部, 助教授 (80127092)
KUSUNOKI Shin. Advance Co., Inst. life Sci., Director
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥8,800,000 (Direct Cost: ¥8,800,000)
Fiscal Year 1987: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1986: ¥7,600,000 (Direct Cost: ¥7,600,000)
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| Keywords | cerebrotendinous xanthomatosis / CTX / cholestanol / figh-performance liquid chromato- graphy / benzoyl derivatization / GC / MS / 高速液体クロマトグラフイー |
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| Research Abstract |
Cerebrotendinous Xanthomatosis (CTX) is a hereditary disorder resulting in an ele- vation in the concentration of cholestanol in serum and tendon xanthomas< A definite diagno- sis of this disease can be made by detemination of the cholestanol concentration. In 1976 we developed an analytical method fro the detemination by GC-MS. For diagnostic purposes, how- ever, a more accurate and easier method is desirable. In the present study, we developed a simultaneous quantification of serum cholestanol and cholesterol by high-performance liquid choromatography (HPLC), which is suitable for the clinical diagnosis of CTX. (methods) Known amount of internal standard was placed in a tube, followed by 100 ul of serum and 1 ml of ethanolic potassium hydroxide. The unsaponifiable material was extracted with hexane after heating for 1 hr at 80 C. The benzoyl chloride reagent was added to the unsaponifiable mate- rial. The benzoyl derivatives were injected into the HPLC system. The column was a SBC-ODS, and the solvent was acetonitril-water-acetic acid. jSteryl benzoates were detected by their adsorbance at 228 nm. Molecular extinction was 12000.
(Results) The alkaline hydrolysis was completed within 60 min. The reaction of benzoyl derivatization was completed in 5 minutes, and the time to analyze one sample was 40 minutes. The total time required after extraction was about 2 hours. The linearities of quantification of cholesterol and cholestanol were ob- served within 0 -3 mg/ml and 0 -100 ug/m1, respectively, This procedure is also applicable to the quantification of sitosterol or campesterol which appear in som inherited diseases.
(Discussion) More than 50 cases of CTX have been diagnosed in our slaboratory, and treatment of this disease by chenodeoxycholic acid administration have been stgarted recently. The pro- sent procedure is suitable for the diagnosis and the follow-up studies of this disease.
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