【作者】 董军；

【导师】 陈保生； 陈文祥；

【作者基本信息】 中国协和医科大学 ， 生物化学， 2008， 博士

【摘要】 准确测定血清脂蛋白胆固醇需首先分离各种脂蛋白。超速离心法是各种脂蛋白的定义方法,同时也是最可靠的脂蛋白分离方法。用超速离心法可以将各种脂蛋白和亚类大致分为高密度脂蛋白（HDL,密度1.063-1012g/mL）、HDL2（1.063-1.125）、HDL3（1.125-1.21）、低密度脂蛋白（LDL,1.006-1.063）、小而密LDL（LDLb,1.044-1.063）、大而轻LDL（LDLa,1.006-1.044）、中间密度脂蛋白（IDL,1.006-1.019）、和一种特殊的脂蛋白（a）[Lp（a）,（1.05-1.11）]。在目前的临床检验领域,IDL和Lp（a）包括在LDL中。由于Lp（a）在HDL和LDL中均有分布,所以用超速离心分离各种脂蛋白及亚类必须首先消除Lp（a）的影响。Lp（a）由一分子类似于LDL的脂蛋白颗粒和载脂蛋白（apo）（a）组成,apo（a）与LDL颗粒中的apoB以二硫键相连接,少量Lp（a）还与其他脂蛋白（主要为含apoB的β-脂蛋白）以非共价键结合。本研究选择用β-巯基乙醇（ME）解离Lp（a）,对ME的浓度、ME解离Lp（a）的有效性、Lp（a）解离对HDL稳定性的影响、脯氨酸的作用等进行试验,发现0.05mol/L ME可以使Lp（a）解离为不含apo（a）的Lp（a）[Lp（a-）]和apo（a）,0.1mol/L脯氨酸可有效消除Lp（a）与其他脂蛋白之间的非共价键结合;解离后的Lp（a-）与LDL密度相近,从而实现HDL的超速离心分离及HDL和LDL[包括IDL和Lp（a）]的测定。进一步的研究发现,解离后Lp（a-）的密度＜1.040,与解离前（1.05-1.11）存在明显的密度分界区域,LDLa和LDLb的密度分界点1.044恰好在此区域,这一特点不仅使超速离心分离测定Lp（a）成为可能,而且还可以分离测定LDLa和LDLb。各种脂蛋白及其亚类胆固醇测定需要精密的胆固醇分析方法。本试验选用市售豆甾醇作内标,用Jones反应氧化胆固醇和内标为胆甾/豆甾-4-烯-3,6-二酮,以提高胆固醇和内标的可分离性及检测灵敏度。所建方法易操作,灵敏度、精密度好,适用于超速离心后脂蛋白组分中低水平胆固醇测定。超速离心高效液相色谱（HPLC）准确测定血清脂蛋白胆固醇的方法如下:血清与含脯氨酸的溴化钠溶液混合,使背景密度为1.006,超速离心分离底部组分（BF）HDL和LDL(BF_1.006);使背景密度为1.044,超速离心分离LDLb、Lp（a）和HDL(BF_1.044)。血清与含ME和脯氨酸的溴化钠溶液混合,使背景密度为1.044,超速离心分离LDLb和HDL(BF_1.044ME);背景密度为1.063,超速离心分离HDL(BF_1.063ME);使背景密度为1.125,超速离心分离HDL3(BF_1.125ME)。不同密度和条件超速离心后的底部组分胆固醇（BFC）测定用HPLC法,计算各脂蛋白组分胆固醇。HDLC=BF_1.063MECLDLC=BF_1.006C-BF_1.063MECLDLaC=BF_1.006C-BF_1.044CLDLbC=BF_1.044MEC-BF_1.063MECHDL2C=BF_1.063MEC-BF_1.125MECHDL3C=BF_1.125MECLp（a）-C=BF_1.044C-BF_1.044MEC超速离心HPLC法测定血清超速离心底部组分胆固醇（HDLC+LDLC）的结果与美国疾病控制与预防中心（CDC）的β-定量法高度相关（r=0.998）;测定HDLC结果与CDC指定比对方法（DCM）高度相关（r=0.998）。本法测定HDLC和LDLC的总CV分别为0.96%～2.07%和0.65%～1.12%;测定HDL2C、HDL3C、LDLaC和LDLbC的总CV分别为0.85-2.66%,0.87-3.21%,0.86-1.11%,和2.59-6.35%。Lp（a）-C测定的总CV为4.42—12.29%。为了降低Lp（a）-C的测定误差,提高灵敏度,设计了两次超速离心直接分离和测定Lp（a）-C的方法。血清溶液在脯氨酸存在下1.044的背景密度超速离心,＞95%的Lp（a）分布在离心管的底部;用含ME的密度液将BF调节密度至1.040,再次超速离心,则解离后的Lp（a-）分布在顶部组分,HPLC测定顶部组分胆固醇即为Lp（a）-C。两次超速离心的方法提高了Lp（a）-C测定的灵敏度和特异性,测定Lp（a）-C的总CV为2.85-4.29%。综上所述,本研究通过研究Lp（a）的解离和解离前后的密度分布建立了血清主要脂蛋白及其亚类胆固醇的测定方法。本方法有以下特点:（1）首次用超速离心分离包括脂蛋白亚类和Lp（a）在内的多种脂蛋白组分,所分离的脂蛋白与应用最广泛的脂蛋白定义一致;（2）物理方法分离脂蛋白,样品处理过程中的脂蛋白胆固醇变化减至最低程度;（3）统一用胆固醇代表各脂蛋白水平,可使各脂蛋白[包括Lp（a）]和亚类进行直接对比;（4）所需样品量小,0.5mL血清可以测定7项主要脂蛋白组分,一次最多可分析20个样品;（5）样品制备简单、方法易操作,精密度良好。本方法具有良好的准确性和实用性,有望作为HDL、LDL及其亚类胆固醇和Lp（a）-C测定的参考方法。更多还原

【Abstract】 The different types of lipoproteins have been defined historically by ultracentrifugation.Lipoproteins can mainly be classified into the following fractions according to their hydrated densities:High density lipoproteins（HDL,1.063-1.12g/mL）, HDL2（1.063-1.125）,HDL3（1.125-1.21）;Low density lipoproteins（LDL,1.006-1.063）, small dense LDL（LDLb,1.044-1.063）,large buoyant LDL（LDLa,1.019-1.044）; intermediate density lipoproteins（IDL,1.006-1.019） and lipoprotein（a）[Lp（a）,1.05-1.11]. LDL cholesterol actually contains the contributions of IDL and Lp（a） cholesterol in current clinical laboratory measurements.In ultracentrifugation,the Lp（a） density range overlaps those of both HDL and LDL.The recovered lipoprotin frations,especially LDLb and HDL2,are likely to be comtaminated with Lp（a）.Lp（a） is formed by joining a lipoprotein that is structurally very similar to LDL to apo（a）.In the Lp（a） particle,apo（a） is covalently linked to apoB by a single disulfide bridge.A small amount of Lp（a） or apo（a） is also noncovalently associated with apoB-rich lipoproteins.2-mercaptoethanol（ME） was used to dissociate Lp（a） in this study.The concentration of ME used,the effectiveness of the dissociation,HDL stability and the effect of proline were investigated.The results showed that 0.05mol/L ME can effectively dissociate Lp（a） into apo（a） and apo（a） depleted Lp（a）[Lp（a-）],and 0.1mol/L proline dissociate the interaction of Lp（a） with apoB-rich lipoproteins.The density range of Lp（a-） is similar to that of LDL,which enables the ultracentrifugation separation of HDL and LDL.Further studies demonstrated that there is a distinct density zone between Lp（a-） （＜1.040） and Lp（a）（1.05-1.11） and the density cut point between LDLa and LDLb （1.044） is coincidentaUy fall into this zone.Based on these findings,an ultracentrifugation method for separation of HDL,HDL2,HDL3,LDL,LDLa,LDLb and Lp（a） were established.A precise method is needed for determination of cholesterol concentrations in separated lipoprotein fractions.A modified HPLC method was developed by using merchandised stigmasterol as an internal standard and the Jones Oxidation for the oxidation of cholesterol and stigmasterol into cholest/stigmast-4-en-3,6-dione.This method is highly precise and can be used for detecting low levels of cholesterol concentrations in lipoprotein fractions.Based on the above investigations,an ultracentdfugation and HPLC method for determinations of serum lipoprotein cholesterol was established.Serum aliquots were centrifuged in the presence of proline and in the presence or absence of mercaptoethanol at densities of 1.125,1.063,1.044 and 1.006 g/mL for the separation of lipoprotein fractions. Cholesterol levels in the ultracentrifugation bottom fractions were analyzed by HPLC. Cholesterol concentrations of lipoprotein fractions were calculated as follows.HDLC = BF_1.063MECLDLC = BF_1.006C - BF_1.063MECLDLaC = BF_1.006C - BF_1.044CLDLbC = BF_1.044MEC - BF_1.063MECHDL2C = BF_1.063MEC - BF_1.125MECHDL3C = BF_1.125MECLp（a）-C = BF_1.044C - BF_1.044MECFor determination of the centrifugation bottom fraction（d=1.006,HDLC+LDLC）, the results provided by ultracentrifugation and HPLC method were highly correlated with those provided by CDC reference method（r=0.998）;For determination of HDLC,the results were highly correlated with those from CDC designated comparison method（DCM） （r=0.998）.The total CVs for the measurement of HDLC and LDLC were 0.96%～2.07% and 0.65%～1.12%;The total CVs for the measurement of HDL2、HDL3、LDLa、LDLb and Lp（a） cholesterol were 0.85%-2.66%,0.87%-3.21%,0.86%-1.11%,2.59%-6.35%and 4.42%-12.29%,respectively.For determination of Lp（a） cholesterol,a more sensitive,two-step ultracentrifugation method was designed.Serum aliquots were centrifuged in the presence of proline at a density of 1.044 g/mL and centrifuged.The bottom fraction,which contains＞95%of Lp（a） particles,was readjusted with a density solution containing ME to a density of 1.040 to cause flotation of Lp（a-） in the next centrifugation.Cholesterol concentration in the top fraction was determined with HPLC.The two-step ultracentrifugation procedure increased the sensitivity and specificity of the method,The total CVs for the measurement of Lp（a）-C was 2.85-4.29%。In conclusion,an ultracentrifugation and HPLC method for determination of cholesterol concentrations in lipoprotein and their subfractions and Lp（a） has been developed.The new method has the following characteristics:（1） Lipoprotein and their subfractions are separated with ultracentrifugation,and the separated lipoproteins are in agreement with the widely used lipoprotein nomenclature;（2） Ultracentrifugation is a physical procedure in which the cholesterol transfers among lipoproteins are minimal.（3） Lipoprotein fractions and Lp（a） levels are all represented by their cholesterol levels,so a direct comparison is possible among these liporoteins.（4） The sample size is low,0.5mL is needed for determinations of 7 lipoprotein fractions.For each analytical run,20 samples can be analyzed.（5） This method is simple,easy to operate and precise,and may be used as a candidate reference method for determination of cholesterol concentrations in HDL, LDL,HDL and LDL subfractions and LP（a）.更多还原