Development of Diagnosis System of Cerebrotendinous Xanthomatosis

脑腱黄瘤病（CTX）是一种遗传性疾病，导致血清中胆固醇浓度升高和肌腱黄瘤。通过测定胆固醇浓度可以明确诊断此病。1976年建立了气相色谱-质谱联用测定方法。然而，为了诊断的目的，更准确和更容易的方法是可取的。在本研究中，我们建立了同时定量血清胆固醇和胆固醇的高效液相色谱（HPLC）方法，适用于CTX的临床诊断。（方法）将已知量的内标放入管中，加入血清100 ul和乙醇氢氧化钾1 ml。不皂化物在80℃下加热1小时后用己烷萃取，不皂化物中加入苯甲酰氯试剂。将苯甲酰衍生物注入高效液相色谱系统。色谱柱为SBC-ODS，溶剂为乙腈-水-乙酸。在228 nm处对苯甲酸乙酯进行吸附检测。分子灭绝是12000。（结果）碱水解在60min内完成。苯甲酰衍生化反应在5分钟内完成，分析一个样品的时间为40分钟。提取后总时间约2小时。在0 ~ 3mg /ml和0 ~ 100ug /m1范围内，胆固醇和胆固醇的定量具有良好的线性关系，该方法也适用于某些遗传性疾病中出现的谷甾醇或油菜甾醇的定量。（讨论）我们实验室已经诊断出50多例CTX，最近开始使用鹅去氧胆酸治疗这种疾病。预送程序适用于本病的诊断和后续研究。

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.