Development of Diagnosis System of Cerebrotendinous Xanthomatosis

脑腱黄瘤病(CTX)是一种遗传性疾病，可引起血清和肌腱黄瘤中胆甾醇浓度的变化，通过测定血清和肌腱中的胆甾醇浓度可对该病作出明确的诊断。1976年，我们建立了一种用气相色谱-质谱法测定的分析方法。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，我们建立了一种同时测定血清胆固醇和胆固醇的高效液相色谱方法，该方法适用于临床诊断环磷酰胺。(方法)将已知量的内标放入试管中，然后加入100ul的血清和1ml的乙醇氢氧化钾。不皂化物在80℃加热1小时后用正己烷提取，在不皂化物中加入苯甲酰氯试剂。将苯甲酰衍生物进样到高效液相色谱系统中。色谱柱为SBC-ODS柱，溶剂为乙腈-水-冰醋酸。在228 nm波长处测定了苯甲酸酯的吸光度。分子消光系数为12000。(结果)碱解反应在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。提取后的总时间约为2小时。胆固醇和胆甾醇的线性范围分别为0~3 mg/m l和0~100 ug/m~1。本方法也适用于某些遗传性疾病中谷甾醇或菜籽甾醇的测定。(讨论)我室已诊断出50多例环磷酰胺中毒，鹅去氧胆酸治疗本病。该方法适用于本病的诊断和随访研究。

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.