【作者】 王萌烨；

【导师】 沈敏； 向平； 沈保华；

【作者基本信息】 复旦大学 ， 法医学， 2008， 硕士

【摘要】 脱氢表雄酮(dehydroepiandrosterone,DHEA)是人体分泌的一种重要生理活性物质,属内源性类固醇激素。类固醇激素(Anabolic steroids),又称甾体同化激素,是近年来使用最频繁与广泛的运动兴奋剂,因此,DHEA等类固醇激素的检测一直是运动兴奋剂检测的热点。尿液与血液是当今兴奋剂检测领域最为普遍采用的检材,但这些检材存在检测时限短等难以克服的缺陷,因此探寻新的检材成为兴奋剂检测研究的热点。毛发具有检测时限长、易于保存、能够反映用药历史等优势,已在滥用药物的检测领域得到了较为广泛的应用。因此,进行毛发中类固醇激素检测的研究,为兴奋剂检测提供技术储备,具有重要的意义。类固醇激素按照来源不同分为两大类:外源性类固醇激素以及内源性类固醇激素。兴奋剂检测在对待这两类激素的策略上有着本质的区别。针对外源性类固醇激素相对比较简单,仅需在检材中检出该类物质即可判为阳性。而内源性激素因人体内存在此类物质,给研究和结果判定带来了复杂性。因此,作为分析方法应用和结果评价的重要环节,必须考察中国人群体内内源性类固醇激素的存在状况,考察中国人群尿液及毛发中内源性类固醇激素的分布情况;探讨通过尿液、毛发分析判断内源性类固醇滥用的可行性,从而为内源性类固醇滥用的分析和评价提供方法和依据。本文对中国人群体内(尿液、毛发)中DHEA及其相关物质的检测、分布及结果评价进行了系统的研究,建立了较为完整的体内DHEA及其相关物质的分析、确认、评价体系。主要包括以下几个方面:一、尿液中DHEA及其相关物质的分析与研究1.建立了液相色谱-串联质谱法(LC-MS/MS)测定尿液中的DEHA及其相关物质的方法。尿样经液-液提取后,采用Cosmosil C18色谱柱,并以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68∶32)为流动相,以三重四极杆串联质谱多反应监测扫描方式对脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素成分进行检测。方法的最低检出限为0.01～10ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可应用于体液中类固醇激素的常规分析。2.考察了中国健康成人尿液中DHEA及其相关内源性类固醇激素的水平,揭示服用DHEA后尿液中DHEA的代谢规律以及对其他相关内源性类固醇激素水平的影响。结果表明志愿者尿液中的睾酮浓度明显低于我国和国外运动员。单剂量服用DHEA后,尿液中DHEA、雄酮和苯胆烷醇酮的浓度有明显的升高,达峰时间分别为:3.86±0.64h、3.43±0.90h和3.57±0.90h。因此可以推断尿液中DHEA等内源性类固醇激素水平因种属、年龄、性别、身体素质等因素存在较大差异。监测滥用外源性的DHEA时,其尿液中的DHEA浓度是最有效的技术指标。二、毛发中DHEA及其相关物质筛选方法的建立1.建立了气相色谱-串联质谱法(GC-MS/MS)测定毛发中的DHEA及其相关类固醇激素的筛选方法。头发经碱水解后液-液提取,利用MSTFA衍生化试剂衍生化后以串联四极杆质谱多反应监测扫描方式对脱氢表雄酮、睾酮、表睾酮、雄酮、苯胆烷醇酮、诺龙、甲基异睾酮、甲基睾酮、去氢甲基睾酮、乙诺酮等10种激素成分进行检测。方法的最低检出限为0.2-5pg/mg,平均回收率为71.0%～110.5%,日内和日间相对标准偏差(RSD)分别小于17%和18%。结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可应用于毛发中类固醇激素的常规分析。2.应用建立方法测定了健康人头发中DHEA及其相关内源性类固醇激素。此外,按照NIST所列方法自行制备了类固醇激素的阳性毛发,应用所建立的方法对阳性毛发中去氢甲基睾酮与乙诺酮进行了测定。三、中国健康人头发中DHEA及其相关类固醇激素的检测与评价1.应用所建立的筛选方法测定了88名不同性别和年龄的中国健康人头发中DHEA及其相关内源性类固醇激素含量。2.考察了不同性别年龄健康中国人头发中DHEA及其相关内源性类固醇激素分布情况,结果表明头发中雄酮、表睾酮、苯胆烷醇酮的含量明显低于DHEA与睾酮的含量;男性头发中睾酮的含量在20-40年龄段达到峰值随后随着年龄的增长下降;男性女性头发中睾酮含量没有明显区别。四、DHEA在豚鼠毛发中的分布研究1.测定了豚鼠毛发中内源性DHEA的含量。取单色、双色或三色毛发豚鼠共22只,剃去背部取样区的毛发,测定空白毛发中DHEA的含量,结果表明豚鼠毛发中内源性DHEA含量与毛发颜色没有明显关系,与人头发中DHEA含量相比较没有明显区别。2.考察了单次注射DHEA后豚鼠毛发中DHEA含量变化过程。选取背部单色、双色或三色毛发豚鼠各6只,给药前剃光背部取样区毛发,以20mg/kg剂量单次腹腔注射DHEA,隔天剃毛,分别包装保存,连续7次,测定DHEA的含量。结果表明毛发中DHEA的含量高峰出现在给药后的第二天。3.考察了多次给药后豚鼠毛发中DHEA含量的变化过程。选取背部黑色毛发豚鼠共18只,分成高、中、低3组。给药前剃光背部取样区毛发,以20mg/kg剂量注射DHEA,连续注射3天,第一次给药后第二天剃毛并隔天取毛,分别包装保存,连续7次,测定毛发中DHEA的含量。结果表明三个剂量组中DHEA含量均在末次给药后第二天达到峰值,毛发中DHEA的含量与给药剂量相关,低剂量组毛发中DHEA含量变化不明显。4.毛发颜色对注射DHEA后豚鼠毛发中DHEA含量的影响,取背部长有不同毛发色块(黑、白、棕)的豚鼠6只,给药前剃光背部取样区毛发,以20mg/kg剂量腹腔注射DHEA,给药后第二天剃毛,测定所得各色毛发中DHEA的含量。结果表明同体豚鼠毛发中DHEA的含量与颜色有关,含量从大到小依次为黑色、棕色、白色。综上所述,本文的创新点及研究的意义如下:1.本文所建立的尿液中DHEA及其相关内源性类固醇激素的定性定量分析方法灵敏、专属性强、为国内首创,该方法的检测限与定量限明显低于其他研究报导结果,总体指标达到国际先进水平。2.首次测定了中国人群毛发中DHEA及其相关内源性类固醇激素的含量,为阳性结果的判定提供了依据。讨论了中国人群不同年龄、性别毛发中内源性类固醇激素含量的分布。为毛发中内源性类固醇激素结果的判定提供了依据。3.首次对DHEA在豚鼠毛发中的分布及时间过程进行了研究,结果表明给于外源性DHEA后黑色毛发中的DHEA结合率较高。并且中国人黑色头发适合毛发中内源性类固醇激素的检测。更多还原

【Abstract】 Dehydroepiandrsterone （DHEA）, a very important biological active prehorone, is a kind of endogenous steroid. In recent years, DHEA and other related steroids have always been widely and frequently used so that the detection of these steroids has become very hot. In this field, although urine and blood are 2 commonly adopted specimens they have many shortcomings that very difficult to be overcame. Recently, hair, a variety of specimen other than blood and urine, has been purposed to drug exposure because hair has a long detection time limit, can be easily conserved and indicate the history of drug exposre. So the study of the determination of steroids in hair which is to provide evidence to help hair become the specimen of anti-doping is of great importance.Anabolic steroids can be devided into 2 types: exdogenous steroids and endogenous steroids. The strategies on the detection of these 2 types of steroids are totally different. To determine exogenous steroids, if the steroids can be detected the results is positive. To determine endogenouse steroids, because the steroids exist in the body the judgement of the result is very diffictult. Before the dtermination of the endogenouse steroids, we must study the conditions of endogenous steroid in human body and discuss the feasibility of deciding endogenous steroids abuse and then provide evidence and methods information for the analysis and evaluation of endogenous steroids abuse.This paper systematically studied the detection, distribution and evaluation of DHEA and related steroids in vivo among Chinese people and established a complete analasys, conformation and eavalation system for DHEA and related steroids. The comments are as below:1. The analysis and study of DHEA and related steroids in urine（1） A method was developed for the determination of endogenous steroids in urine with methyltestosterone as internal standard. After liquid-liquid extraction, the urine sample was chromatographed on a Cosmosil C₁₈ column with a mixture of methanol and ammonium acetate-formic acid （68:32, v/v） as mobile phase, then detected with liquid chromatography-tandem mass Spectrometry （LC-MS/MS） system with electrospray ionization （ESI） in multi-reaction monitoring （MRM） mode. The detection limits ranged from 0.01 ng/mL （for testosterone and epitestosterone） to 10 ng/mL （for androsterone and etiocholanolone）. The recoveries ranged from 96.7% to 106.5% and intra- and inter-day precisions （measured as relative standard deviations） were less than 7% and 11%, respectively. With simple and fast sample preparation, the method was sensitive and specific for simultaneous determination of these 5 kinds of endogenous steroids in urine. The method has been successfully applied in pharmacokinetic study and was thus a potential alternative for gas chromatography-mass Spectrometry （GC-MS） based procedures in routine analysis of endogenous steroids such as DHEA in human urine.（2） Studied the level of DHEA in Chinese humans’urine and revealed the influence of oral DHEA to the metabolism of DHEA in urine and the level of other endogenous steroids in rine. The results showed that the level of testosterone in Chinese humans’urine is lower than Chinese and foreign athletes. After single oral administration of DHEA, the levels of DHEA, androsterone, etiocholanolone raised and the peak of the level were 3.86±0.64h、3.43±0.90h and 3.57±0.90h separately. Through these results we concluded that the levels of DHEA and other related endogenous steroids varied because of ethnic group, age, gender and physical quality. The concentration of DHEA in urine could be the most effective criteria for the determination of DHEA administration.2. The establish of the screening method of DHEA and related steroids in hair（1） A method was developed for the screening of DHEA and related steroids in hair with d3-testosterone as internal standard. After liquid-liquid extraction, the hair sample was derivatived by MSTFA, then detected with gas chromatography-tandem mass Spectrometry （GC-MS/MS） system in MRM mode. The detection limits ranged from 0.2 pg/mg to 5 pg/mg . The recoveries ranged from 71.0% to 110.5% and intra- and inter-day precisions （measured as relative standard deviations） were less than 17% and 18%, respectively. With simple and fast sample preparation, the method was sensitive and specific for simultaneous determination of these 10 steroids in hair. The method could be a potential method in routine analysis of steroids such as DHEA in human hair.（2） Determined DHEA and other related endogenous steroids in healthy Chinese people’s hair. After making positive hair following the instruction published by NIST, we determined methandienone and norethandrolone in the positive hair.3. The determination and evaluation of DHEA and related steroids in Chinese healthy people’s hair.（1） Determined the concentration of DHEA and other related steroids in 88 Chinese healthy people’s hair divided by different age group and gender.（2） Studied distribution condition of DHEA and related endogenous steroids in 88 healthy Chinese people’hair divided by different age group and gender. The results showed that the concentration of androsterone, epitestosterone, etiocholanolone was lower than that of DHEA and testosterone, the concentration of testosterone in men’s hair reached peak in the age of 20 to 40 and there was no obvious difference between men and women’s hair.4. The study of distribution of DHEA in ginea pig’s hair（1） Determined the concentration of endogenous DHEA in ginea pig’s hair. Divided 22 ginea pigs into 3 groups: single color, double colors and triple colors. Determined the concentration of endogenous DHEA after taking the hair on genia pigs’ back. The result showed that there was no significant difference between different color and the concentration of DHEA was as high as that of humans’ hair.（2） Studied the changing process of DHEA concentration in genia pigs’ hair after single administration. Divided 6 ginea pigs into 3 groups: single color, double colors and triple colors. After shaving out the hair on genia pigs’ back give a dose of 20mg/kg DHEA through intraperitoneal injection and then collected the hair every 2 days which lasted 7 times. Determined the concentration of DHEA after taking the hair on genia pigs’ back. The result showed that the concentration reached the peak on the second day after the administration.（3） Determined the changing process of DHEA concentration in genia pigs’hair after multiple administration. Divided 18 ginea pigs into 3 groups through the dose. After shaving out the hair on genia pigs’ back give doses of 20mg/kg, 4mg/kg and 0.8 mg/kg DHEA through intraperitoneal injection 1 time/day（3 days）. Took the hair on the second day after the administration and then took every 2 days which lasted 7 times. Determined the concentration of DHEA after taking the hair on genia pigs’ back. The result showed that the changing process of the 3 groups were nearly the same and the changing of DHEA was not significant in the low dose group.（4） Sudied the influce of hair color to the DHEA concentration after DHEA administration. Chose 6 genia pigs with 3 different hair color on the back. After shaving out the hair on the back gave a dose of 20mg/kg through intraperitoneal injection. Took the hair every 2 days which lasted 7 times and determined the DHEA concention in the hair. The result showed that the DHEA concentration in the hair changed because of the hair color and the concentrations were black, brown and white from high to low.Innovation points and significance are as below:1. The quatitation and determination method for DHEA and related steroids in urine were sensitive and specific. The LOD and LOQ were significantly low than the other study and overall indicator reached advanced international level.2. First determined DHEA and related steroids in Chinese people’s hair and provided evidence for the judgement of positive case. Discussed the distribution of endogenous steroids in Chinses people’s hair among different age and gendor. Provided evidence for the detection of endogenous steroids in the field of anti-doping.3. First studied the distribution and changing process of DHEA in genia pigs’s hair. The results showed that the exogenous DHEA binded with black hair more easily than the other hair color and Chines people’s hair were suitable for the detection of endogenous steroids.更多还原