摘要
一研究背景
随着人民生活水平的提高以及人口老龄化进展的加快,近20年来我国心脑血管疾病发病率与死亡率逐年升高。冠心病(CAD)是由冠状动脉粥样硬化引起的心肌缺血性疾病,动脉粥样硬化(AS)是CAD的病理基础,脂质浸润学说贯穿了AS的形成、进展及恶化,并与其他学说密切相关,血脂异常是AS发生和发展的最重要因素之一。大量流行病学资料显示,AS的严重程度随血清胆固醇水平的升高呈正相关,血清胆固醇的浓度与冠心病的死亡率呈正相关。低密度脂蛋白胆固醇(LDL-C)升高导致的血脂异常是AS病理改变基础之一,AS,的严重程度与LDL-C水平的升高呈线性加重,LDL-C水平与各种心脑血管事件的发生率呈正相关。临床治疗上,我们采用他汀类等药物降低LDL-C水平,可显著减少心脑血管疾病的发生,降低LDL-C能让病人在临床治疗中获益。但是,尽管LDL-C水平达到指南要求甚至低于指南要求,但患者心脑血管事件发生率也只能降低30%左右,依然残留70%左右心血管事件发生率无法避免。
高密度脂蛋白胆固醇(HDL-C)是另外一种重要的脂蛋白,HDL具有心血管保护作用,大量流行病学资料表明,血清HDL-C浓度与CAD发病危险呈独立的负相关；Framingham研究发现,血清HDL-C<35mg/dl者的CAD死亡率为血清HDL>55mg/dl者的4.1倍；血清HDL-C水平每升高1mg/d1,男性患者CAD的危险性即降低2%,女性降低3%。以上结果均提示,HDL是AS的保护因素之一,升高HDL-C水平是抗AS的靶点之一。HDL通过多种机制发挥抗AS的功效,包括对逆胆固醇转运(Reverse Cholesterol Transport, RCT)、抗氧化、抗炎、改善内皮细胞功能失调、抗血栓及促纤溶等。
然而,越来越多的研究发现升高HDL-C水平并未达到人们预期的效果,CETP抑制剂torcetrapib尽管大幅度升高HDL-C水平,但torcetrapib组的全因死亡人数较阿托伐他汀组多,该研究不得不提前终止。Gomarasch等发现在载脂蛋白AI(ApoA-I)转变为ApoA-I Milano人群中,HDL-C的水平低,但其CAD的发生率也低且明显长寿。肝脏清道夫受体BI(SR-BI)高度表达小鼠,虽然降低了HDL-C水平,却具有显著的抗AS功能。因此HDL-C与AS的关系比我们想像中更加复杂,我们临床工作者及科研人员应重新审视HDL-C水平的意义,不单单需要关注HDL-C的水平,更需要关注HDL的功能对AS的影响。
正常HDL功能的HDL才能发挥抗AS作用,而功能异常的HDL甚至有促AS进展的作用。HDL的多面性,与其结构复杂密切相关,HDL包含着多种酶和脂蛋白,其代谢途径也受多种酶的影响,其中任何一个脂蛋白性质的改变、酶的活性或者量的改变均能影响HDL的正常功能。RCT是HDL抗AS的重要的功能之一,RCT是一个复杂的过程,外周组织(包括动脉壁)游离胆固醇在多种酶的作用下与血清中ApoA-I结合后逐渐成为成熟的富含胆固醇的大颗粒HDL,其与肝脏SR-BI受体结合并被肝脏获取,最后HDL内的胆固醇通过肝细胞代谢,最终经胆汁排泄,从而使集聚在血管内皮下胆固醇排泄至体外,达到缓解甚至逆转AS的作用。RCT足由多种酶、多种受体、多种脂蛋白共同参与的复杂过程,ApoA-I,LCAT和CETP这两个酶在RCT过程中起着极其重要。
抗炎抗氧化功能是HDL的另一个重要的功能。正常功能的HDL颗粒具有强大的抗氧化能力,一方面HDL含有多种抗炎抗氧化酶(如：PON1、ApoA-I),可以中和已形成的Ox-LDL的毒性反应；另一方面,HDL可以抑制氧化因子和炎症因子(如：MPO、LPO、SOD)的毒性作用,减少机体的炎症瀑布链反应,从而延缓AS的进展。正常情况下,ROS的产生和清除保持平衡状态,在动脉粥样硬化情况下,氧化产物生成大于抗氧化能力产生氧化应激,将导致血管的持续损伤,从而加剧动脉粥样硬化进展。功能异常的HDL容易形成Ox-HDL,而被氧化形成的Ox-HDL不单不具有抗氧化的能力,甚至促进Ox-LDL的形成。
内皮的保护作用是HDL一个重要的功能。一方面HDL抑制单核细胞粘附和迁移到血管壁,刺激内皮细胞修复和增殖,抑制生长因子诱导的血管平滑肌细胞的增生；另一方面,HDL可以促进NOS催化内皮细胞内L-精氨酸产生,促进内皮细胞生成NO；升高血管内皮功能,还可以抑制血小板和白细胞在受损内膜的聚集和粘附,防止内皮功能受损。HDL可以通过多种途径促进eNOS的表达,减少抑制eNOS生成的因素,起到保护内皮功能的作用。
脂蛋白相关磷脂酶A2(Lp-PLA2)是新近发现的与动脉粥样硬化密切相关,可将Ox-LDL水解成大量的溶血卵磷脂和游离氧化脂肪酸,这些促进动脉粥样硬化作用产物进一步活化粒细胞和Lp-PLA2,正常功能的HDL能结合Lp-PLA2,减少机体游离Lp-PLA2量,从而减少血栓的形成。
Hcy升高同样也会引起多种疾病的发生,尤其增加心脑血管事件的发生的风险。同型半胱氨酸的合成和代谢保持着动态平衡,Hcy高于15.0μmol/L称为高同型半胱氨酸血症(hyperhomocy steinemia,高Hcy)。Hhcy是除高血压、高脂血症、吸烟、肥胖、心脑血管病家族史的一个新的独立的AS的危险因素,并且Hcy水平和血管危险之间呈正性相关关系。血浆Hcy每升高5μmol/L,缺血性心脏病发生率增加32%,卒中发生率增加59%,血浆Hcy每降低3μmol/L,可使缺血性心脏病风险降低11-20%,脑卒中风险降低15-33%。血浆总Hcy水平每升高5μmol/L则冠心病危险性男性增加60%,女性增加80%,相对危险性男性为1.6,女性为1.8,相当于总胆固醇每升高20mg/dL的危险性,因此高Hcy是冠心病的一个独立危险因素。
脂质学说是动脉硬化的基础,目前国内外对Hcy与脂质学说的关系的研究主要集中在：1、Hey可以影响脂质代谢,使LDL代谢减慢,最终导致机体LDL-C水平的升高。2、Hey致LDL-C沉积作用,Hey可作用于低密度脂蛋白导致其载脂蛋白B的游离氨基巯基化,形成Hey与LDL复合物,这种被巯基化修饰的LDL可以不受负反馈抑制被巨噬细胞吞噬,胞内降解增加,引起胞内胆固醇堆积。3、Hcy致脂质氧化致炎作用：Hcy含自由巯基,可以自身氧化成Hcy混合性二硫化物和Hcy硫内酯,同时产生大量过氧化物,引起蛋白质损伤,清除氧自由基的酶活性降低,使得细胞和组织发生炎症反应,同时产生大量的Ox-LDL,加剧AS进展。
HDL是心血管的保护因素之一,但其与Hcy的关系目前尚无明确的定论。Barter对近年HDL与HCY的关系进行综述发现,既有研究认为Hcy能影响HDL的功能,又有部分研究发现Hcy不影响HDL的功能。然而,越来越多的研究发现,Hcy可以通过影响HDL的某些酶的活性而影响HDL的功能,HCY通过影响PON1酶的活性,而影响HDL抗炎抗氧化功能。Clifford研究发现,HCY与CETP酶的活性密切相关,Hcy浓度升高可以影响CETP酶的浓度,而CETP是HDL代谢过程中的重要酶之一。然而HDL功能包括RCT功能、抗炎、抗氧化、维持细胞内皮稳定等功能,PON1和CETP仅仅是HDL众多功能酶中的一个,因此PON1活性和CETP酶并不能反映HDL的整体功能。Hcy通过影响PON1活性和CETP而调节HDL功能仅是其作用靶点之一,高Hcy可能通过更多的机制影响HDL功能。
二研究目的
本研究通过研究探讨Hcy和MTHFR基因与正常HDL-C患者冠状动脉硬化的关系,探讨Hcy与冠心病患者冠脉病变程度的相关性。通过检测高Hcy患者和对照组患者(两组患者HDL水平均在正常范围)HDL颗粒中的Apo-AI,LCAf、 CETP酶的含量,探讨高Hcy对HDL的RCT功能的影响。通过检测HDL颗粒中PON1、MPO活性,Lp-PLA2、LPO、SOD和eNOS水平,探讨高Hcy对HDL抗炎抗氧化能力的影响,从而为预防和治疗冠状动脉粥样硬化提供新靶点。
三实验方法
1.1研究患者入选标准、分组和排除标准
本研究随机入选2011年1月～2012年6月佛山市顺德第一人民医院住院患者123例,入选患者全部采集吸烟史、高血压病史、糖尿病史、冠心病家族史和其他既往病史。根据根据美国心脏协会(AHA)推荐,正常空腹血浆同型半胱氨酸水平高于15.0μmol/L,则可诊断为高同型半胱氨酸血症,根据是否存在Hcy升高分为正常对照组和高Hcy组。两组入选人群的HDL-C均正常范围(1.16μmmol/L 1.2血样的采集、保存和生化指标测定
早晨8：00前抽取受试者空腹静脉血,部分静置离心放-80℃保存。部分血清及全血立即送顺德第一人民医院检验科用日立7600-210ISE型全自动生化分析仪测定测定：TC、TG、HDL-C、LDL-C、Apo-AI、Apo-B、FBS、PBS、Cr、 BUN、hs-CRP。
1.3血浆Hcy的测定
采用Agilent1100液相色谱仪,分别由不同的两人在同一天来测定,准确性和重复性均符合要求。批内变异为3.7%,批间变异为4.1%；质控样品测定结果偏差低水平点小于20%,中、高水平点测定结果偏差小于15%；样本两次测定值误差不超过15%；如果误差大于15%则重新测定,直到误差小于15%。
1.5MTHFR基因型测定
抽提空腹静脉血离心后得到的白细胞中的基因组DNA后,MTHFR C677T多态性位点基因型检测采用PCR-RFLP方法检测MTHFR基因分型,再通过2%琼脂糖凝胶电泳分开得到不同长度的片段。
1.6冠脉病变情况
所有患者均利用Philip H3000造影机进行冠状动脉造影采集影像,冠脉病变情况由2位专科医师仔细分析,意见有不一致时由第3人核实确定。按病变累及左前降支,左回旋支与右冠状动脉的支数,分为单支,双支和多支病变组；根据美国心脏协会规定的冠状动脉血管图像记录分段评价标准,采用Gensini积分系统对各支血管病变程度进行定量评定。
1.7高密度脂蛋白的分离
采用密度梯度离心法分离高密度脂蛋白,BCA法测定其浓度。
1.8HDL逆转运酶的检测
采用酶联免疫吸附剂盒测定HDL颗粒中的LCAT和CETP酶的量。
1.9HDL抗炎抗氧化功能的测定
采用酶联免疫吸附剂盒测定HDL颗粒中的eNOS和Lp-PLA2酶的量。采用分光光度计法测定PON1、MPO酶的活性和LPO、SOD的量。
1.10统计学分析
所有计量资料均以均数±标准差(x±s)表示,并采用SPSS13.0统计软件进行统计分析,计量资料采用两样本t检验,计量等级资料采用两独立样本秩和检验,计数资料采用例(%)表示,采用卡方检验,最后进行两等级变量间的Spearman相关性分析、两计数资料进行Pearson相关性分析和多元线性回归方法；P≤0.05为差异有统计学意义。
四结果
人口学特点比较
与对照组相比(12.4±1.58mmol/L),高Hcy组(18.99±2.71mmol/L)患者的血清Hcy浓度明显升高(P=0.000),差异具有统计学意义。比较对照组和高Hcy组患者的年龄(χ2=0.407,P=0.685)、性别分布情况(χ2=0.003,P=0.959)、高血压患者(χ2=0.421,P=0.517)、糖尿病患者(χ2=0.778,P=0.378),吸烟患者(χ2=1.014,P=0.314)差异无统计学意义。
两组患者的实验室指标比较
比较对照组和高Hcy组患者的TC(t=0.534,P=0.595)、TG(t=0.457,P=0.649)、HDL-C (t=1.347, P=0.181)、LDL-C (t=0.650, P=0.517)、Apo-B (t=1.426,P=0.147)、空腹血糖(t=0.687,P=0.494)、餐后2h血糖(t=0.484,P=0.630)、Cr(t=0.301,P=0.764)、BUN(t=0.443,P=0.659)、ALT(t=0.716,P=0.476)、AST(t=1.725,P=0.088)间差异无统计学意义。入选两组患者生化指标情况一致,具有可比性。
两组患者冠心病冠脉造影结果的比较
对照组相冠脉多数无明显狭窄,或以单支病变为主,而高Hcy组患者冠脉病变程度严重,以单支和双支病变为主,对两组患者进行卡方检验发现,两组患者冠脉病变情况有差异,同时差异具有统计学意义(χ2=17.731,P=0.000)。再进行冠脉评分发现,高Hcy组患者冠脉积分情况较对照组升高,差异具有统计学意义(t=4.222,P=0.000)。
两组患者MTHFR基因分型比较
与对照组MTHFR基因分型以纯合型为主相比,高Hcy组患者MTHFR基因分型以杂合型的CT型为主,同时存在纯变异型TT型2例,进行卡方检验发现,两组患者分型有差异,同时差异具有统计学意义(χ2=7.463,P=0.024)。
MTHFR基因分型与血浆Hcy水平及冠脉病变严重程度相关性分析
血浆Hcy水平与MTHFR基因分型呈正相关(r=0.258,P=0.014),但相关关系并不密切。冠脉病变程度(r=0.600,P=0.000)和冠脉积分(r=0.621,P=0.000)与MTHFR基因分型呈正相关。Hcy水平与冠脉积分存在正相关(r=0.641,P=0.000)；Hcy水平与冠脉病变程度也呈正相关(r=0.438,P=0.000),但相关关系并不密切。最后,我们对冠脉积分与其他指标进行多元线性回归分析,以冠脉积分为因变量,其他变量为自变量,模型纳入MTHFR分型基因、HCY、TC、 Apo-AI水平模型有统计学意义(F=45.923,P=0.000)。MTHFR基因分型(t=7.851,P=0.000), HCY(t=5.805, P=0.000)、TC (t=2.748, P=0.007)、Apo-AI (t=-2.253,P=0.027)水平对冠脉积分有显著影响,差异具有统计学意义。
高Hcy对Apo-AI的影响
与对照组比较(1.27±0.12mmol/L),高Hcy组患者Apo-AI明显降低(1.21±0.13mmol/L)显著降低,差异均有统计学意义(t=2.28,P=0.025)。
高Hcy对LCAT酶的影响
与对照组比较(1029.02±133.88U/mg),高Hcy组患者LCAT酶(913.20±117.42U/mg)显著降低,差异均有统计学意义(t=4.318,P=0.000)。
高Hcy对CETP酶的影响
我们对HDL的重要代谢酶CETP检测发现,高Hcy组(26.33±4.13ug/mg)患者CETP酶比对照组(34.70±4.72ug/mg)明显降低,差异均有统计学意义(t=8.854,P=0.000)。
高Hcy对PON1的影响
与对照组比较(450.55±48.55U/ml),高Hcy组患者PON1活性明显降低(206.48±75.02U/ml)显著降低,差异均有统计学意义(t=17.087,P=0.000)。
高Hcy对MPO酶的影响
我们对MPO活性检测发现,高Hcy组(5.02+2.02U/L)患者MPO活性比对照组(3.15±1.49U/L)明显升高,差异均有统计学意义(t=4.737,P=0.000)。
高Hcy对Lp-PLA2酶的影响
与对照组比较(37.35±8.85ng/mg),高Hcy组患者Lp-PLA2酶明显降低(31.80±6.91ng/mg)显著降低,差异均有统计学意义(t=3.326,P=0.001)。
高Hcy对LPO酶的影响
与对照组比较(0.82±0.09umol/gprot),高Hcy组患者LPO量明显降低(0.93±0.9umol/gprot)显著升高,差异均有统计学意义(t=5.583,P=0.000)。
高Hcy对SOD和eNOS的影响
与对照组比较(190.32±44.57U/m1),高Hcy组患者SOD活性(204.30±49.37U/ml)升高,但差异无统计学意义(t=1.360,P=0.177)。我们再对eNOS活性检测发现,高Hcy组(5.02±2.02ug/mg)患者eNOS活性比对照组(3.15±1.49ug/mg)升高,但两者差异均无统计学意义(t=1.338,P=0.184)。
五结论
1.高Hcy组患者冠脉病变程度严重,冠脉积分情况较对照组升高,Hcy水平与冠脉积分存在正相关,提示高Hcy可能是冠心病的一独立危险因素。
2.高Hcy组患者MTHFR基因分型以杂合型的CT型为主,同时存在少量纯变异型TT型；冠脉病变程度和冠脉积分呈与MTHFR基因分型呈正相关。
3.高Hey可能通过降低HDL颗粒中Apo-AI量,降低LCAT、CETP酶量抑制HDL的RCT功能,促进动脉粥样硬化的进展。
4.高Hcy通过降低PON1活性,减少HDL颗粒中Lp-PLA2量抑制HDL的抗炎抗氧化能力,促进动脉粥样硬化的进展。
5.高Hcy通过升高MPO活性,增加HDL颗粒中LPO量而降低HDL的抗炎抗氧化功能,从而促进动脉粥样硬化的进展。
6.高Hcy并不影响HDL颗粒中SOD和eNOS水平,该结果尚需进一步探讨。
7.综上所述,高Hcy组患者冠脉病变程度严重,Hcy水平与冠脉积分呈正相关,提示高Hcy可能是冠心病的一独立危险因素,机制可能与Hcy患者HDL抗AS功能减弱(失功能)有关：高Hcy患者HDL颗粒中Apo-AI、 LCAT、CETP含量减少,抗炎抗氧化功能减弱,导致HDL的功能下降,促进动脉粥样硬化的进展。降低血浆Hey水平,可能改善HDL的功能,降低Hcy可能成为治疗动脉粥样硬化的靶点之一。
Study Background
With the improvement of people's life quality and the rapidly aging population, the morbidity and mortality of cardiovascular and cerebrovascular diseases are increasing fast. Atherosclerosis (AS) is important pathological basis of coronary heart disease a myocardial ischemic disease cause by Coronary atherosclerosis. Dyslipidemia is one of the most important factors for atherosclerosis and it come with the formation, progress and deterioration of atherosclerosis. Almost all epidemiological studies had demonstrated a positive relationship between total cholesterol and low-density lipoprotein cholesterol whit mortality of cardiovascular. LDL-C level is the most important pathological changes of atherosclerotic risk factor. Patients can benefit from lowering LDL-C treatment through Statins. We found that patients'LDL-C level receiver desired level can reduce cardiovascular events by30%, but remain70%cardiovascular events would be happened. So we believe that high density lipoprotein (HDL) was a therapy target in atherosclerosis prevention.
High-density lipoprotein cholesterol is another important lipoprotein which have Atheroprotective property. Almost all epidemiological studies had demonstrate an inverse relationship between plasma levels of HDL-C and cardiovascular atherosclerotic disease. The Framingham study found people of HDL-C levels of <35mg/dl have4.1times mortality rate than people of HDL-C levels of>55mg/dl Franceschini showed that for every1mg/dl increase in HDL-C, the predicted incidence of coronary events decreases by2%in men and3%in women. The major cardiovascular protective effects of HDL function may be attributed to its role in reverse cholesterol transport (RCT), anti-oxidant and anti-inflammation and so on.
However, several lines of evidence indicate that the relationship between HDL and CAD risk is more complex and extends beyond the serum HDL-C levels. Torcetrapib, a potent CETP inhibitor, markedly increased the plasma concentration of HDL-C, but the risks of deaths and cardiac events in patients receiving tocetrapib had been increased simultaneously. The Milano people who carry the apolipoprotein A-I Milano mutant have very low serum HDL-C level while show very low incidence of CAD. Our results support the theory that not all HDL possess atheroprotective properties. Some HDL is dysfunctional or pro-inflammatory. Surem HDL-C level is not equivalent to HDL function.
The proposed atheroprotective properties of HDL are multifaceted, including Reverse Cholesterol Transport and cholesterol efflux capacity, anti-oxidative and anti-inflammatory activities. The most important theory revolves around the role of HDL in macrophage reverse cholesterol rransport (RCT), in which excess cholesterol is effluxed to HDL and ultimately returned to the liver for metabolism by receptor of ATP binding cassette transporter A1(ABCA1) and Scavenger receptor class B type I (SR-BI).
Structural modification and composition alteration of HDL may lead to HDL loss of normal biological function, and HDL-C is normal which still failed to inhibit atherosclerosis. The most important theory revolved around the role of HDL function is RCT, in which excess cholesterol effluxes to HDL and ultimately returned to the liver for metabolism. The process of RCT is extremely complicated:At first, cholesterol efflux from macrophages and then lipid-poor ApoA-I quickly acquires it via the ABCA1transporter. Lipidation of the lipid-poor ApoA-I and cholesterol complex generates nascent (pre-β) HDL. Subsequently, lecithin cholesterol acyl transferase (LCAT) mediated cholesterol esterification generates small HDL3particles; small HDL3can be converted to large mature HDL2in turn upon CETP [20]. At last, these mature HDL2transfer its cholesterol to the liver directly via SR-BI and subsequently excrete cholesterol through the bile. The process of RCT is extremely complex and ApoA-I, LCAT and CETP play an important role in this process.
HDL has anti-inflammatory and anti-antioxidant property, which play an important role in AS protecting. HDL plays an important role in protecting against LDL oxidative modification, the enzyme PON1and ApoA-I contribute the key role to the antioxidative effects of HDL. MPO, LPO, SOD can change the structure of HDL which would oxidative modification HDL in to ox-HDL, and thus may reduce property of anti-inflammatory and anti-antioxidant in HDL, even to.produce more ox-LDL.
The role of endothelium protection is an important function of HDL. On one hand, HDL inhibits monocyte adhesion and migration to the vessel wall; stimulate the repair and proliferation of endothelial cells, inhibited proliferation of vascufar smooth muscle cells by growth factor. On the other hand, HDL can promote endothelial cells producing the L-arginine and NO by-the NOS catalytic to elevated vascular endothelial function, can also prevent endothelial dysfunction by inhibit the aggregation and adhesion of platelets. HDL play a important role in the protecting endothelial function can by eNOS.
Lipoprotein-associated phospholipase A2(LP-PLA2) is a newly discovered closely related with atherosclerosis. It hydrolyzed ox-LDL into a large number of lysolecithin and free oxidation of fatty acids. The function HDL can bind Lp-PLA2; reduce the free amount of Lp-PLA2and the formation of thrombus.
High homocysteine causes many kinds of diseases, especially increase the risk of cardiovascular and cerebrovascular events. The synthesis and metabolism of homocysteine are maintaining homeostasis, higher than15.0μmol/L called high hyperhomocysteinemia (HHcy). Hhcy is a new independent risk factor of AS including hypertension, hyperlipidemia, smoking, obesity, cardiovascular and cerebrovascular disease, which was a positive relationship between Hcy levels and cardiovascular risk. The plasma Hcy escalate5umol/L can increase ischemic heart disease by32%and stroke by59%; Plasma Hcy reduced3umol/L, the risk of ischemic heart disease can be reduced by11to20%, and the risk of stroke reduced by15-33%. Total plasma Hey level is increased by5umol/L is equivalent to the the total cholesterol each elevated a20mg/dL risk, and therefore to be an independent risk factor for coronary heart disease.
Dyslipidemia is the basis of atherosclerosis. Relationship of Hey and dyslipidemia are unclear. HHcy slows down LDL metabolism and elevated levels of LDL-C. Hey thiolated low density lipoprotein into Hcy-LDL complexes, which cannot be macrophage phagocytosis, increased intracellular degradation, caused by the intracellular accumulation of cholesterol. HHcy can autoxidation to produce large amounts of peroxide, causing protein damage, which produce large amounts of Ox-LDL, exacerbated atherosclerosis progression.
High-density lipoprotein is cardiovascular protective factor, but its relationship with Hey is no clear now. A research found that Oral folic acid to reduce high Hey can be improved PON1activity in the HDL particles, thereby reducing the content of diabetes Ox-LDL and alleviate the progression of atherosclerosis. PON1is one of the HDL enzymes; PON1can not reflect the HDL function. HHcy may affect HDL function through more mechanism.
Objectives
The study was to investigate the relationship between HCY and MTHFR gene and atherosclerosis in in Patients with Medium HDL level, explore the relevance of HCY with coronary artery disease severity. We investigated the impact of HHcy to the RCT function of HDL by detecting the amount of Apo-AI, of LCAT, CETP in HDL particles. We also investigated the anti-inflammatory and anti-antioxidant function of HDL by detecting the PONl and MPO activity, the amount of Apo-AI, of LCAT, CETP in HDL particles.
Methods
1. Patient inclusion criteria, grouping and exclusion criteria
We collect the patient's history of smoking, hypertension, diabetes, family history of coronary heart disease and other past medical history. We divided patients into two groups according HCY levels the HHcy group was defined HCY level above15.0μmol/L and the control group was below15.0μmol/L (guideline of ACC). Both two group HDL level was normal (40mg/dL 2. Serum lipid analysis
Blood lipid analysis was performed at the beginning of the experiment. Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (HDL-C) concentrations were measured by using an automated biochemical analyzer.
3. HCY analysis
HCY analyses were using Agilentl100liquid chromatography, respectively determine by two people in the same day. Of variation of3.7%, inter-assay variation was4.1%; deviation of measurement results of the quality control samples of low-level point of less than20%deviation of the measurement results, a high level point of less than15%; the sample twice determination error does not exceed15%; if the error is greater than15%is re-measured, until the error is less than15%.
4. MTHFR gene analysis
MTHFR was test by PCR-RFLP, and through2%agarose gel electrophoresis to separate the different length fragments
5. Coronary lesions
Coronary angiography was carry by Philip H3000. Coronary lesions were divided into single, double and multi-vessel disease. Gensini score system quantitates assessment of severity of vessel:narrow <25%1score,25%-49%2score,50%-74%4score,75%-90%8score,91%-99%16score,100%32score.
6. The separation of high-density lipoprotein
HDL separated by density gradient centrifugation. BCA assay was use to analysis the HDL particles concentration
7. Assessment of the RCT function of HDL
LCAT and CETP were assayed using Elisa.
8. Assessment of the anti-inflammatory and anti-antioxidant function of HDL
PON1, MPO, LPO, SOD in HDL particles was assayed using kits according to the manufacturer's instructions. eNOS and Lp-PLA2were assayed using Elisa.
Results
1.There were no differences in Age(χ2=0.407, P=0.685), sex (χ2=0.003, P=0.959), hypertension(χ2=0.421,P=0.517), diabetes(χ2=0.778, P=0.378), smoking (χ2=1.014, P=0.314), TC (t=0.534, P=0.595)、TG (t=0.457, P=0.649) HDL-C (t=1.347, P=0.181)、LDL-C (t=0.650, P=0.517)、Apo-B (t=1.426, P=0.147)、FBG (t=0.687, P=0.494)、PBG (t=0.484, P=0.630)、Cr (t=0.301, P=0.764)、BUN (t=0.443, P=0.659)、ALT (t=0.716, P=0.476)、AST (t=1.725, P=0.088) in two group,
2. The Coronary lesions were Serious in HHCY group than control group (χ2=17.731, P=0.000).The Gensini score was higher in HHCY group than control group (t=4.222, P=0.000)
3. There were significant differences in two group in MTHFR gene (χ2=7.463, P=0.024). HCY levels were positively correlated with MTHFR (r=0.258, P=0.014), Coronary lesions(r=0.438, P=0.000) and Gensini score(r=0.641, P=0.000). MTHFR gene were positively correlated with Coronary lesions(r=0.600, P=0.000)and Gensini score(r=0.621, P=0.000).Finally, we take multiple linear regression analysis our data, we found that Gensini score were closed correlated with MTHFR gene (t=7.851, P=0.000), HCY (t=5.805, P=0.000)、TC (t=2.748, P=0.007)、Apo-AI (t=-2.253, P=0.027), the model was meaning full (F=45.923, P=0.000)
4. Conpared with control group, Apo-AI (1.27±0.12mmol/L vs1.21±0.13mmol/L,P=0.025), LCAT (1029.02Q133.88U/mg vs913.20±117.42U/mg, P=0.000) and CETP (34.70±4.72ug/mg vs26.33±4.13ug/mg,P=0.000) was significantly lower in HHCY group.
5. Conpared with control group, PON1(450.55±48.55U/ml vs206.48±75.02U/ml, P=0.000) and Lp-LPA2(37.35±8.85ng/mg vs31.80±6.91ng/mg, P=0.001) was significantly lower in HHCY group.
6. Conpared with control group, MPO (3.15±1.49U/L vs5.02±2.02U/L, P=0.000) and LPO (0.82±0.09umol/gprot vs0.93±0.9umol/gprot, P=0.000) was significantly higher in HHCY group.
7. There were no differences in eNOS and SOD levels.
Conclusions
1. HHCY group coronary lesions were serious than control group, and Gensini score were higher than control group. The HCY level was positive correlated with Gensini score.
2. MTHFR gene in HHCY group was significant differences with control group. The MTHFR gene was positive correlated with Gensini score and coronary lesions. High Hcy may be an independent risk factor for coronary heart disease.
3. HHCY may reduced the RCT function of HDL through decreased. Apo-AI, LCAT, CETP in HDL particles.
4. HHCY reduced the anti-inflammatory and anti-antioxidant function of HDL through decreased PON1and Lp-PLA2in HDL particles.
5. HHCY reduced the anti-inflammatory and anti-antioxidant function of HDL through increased MPO and LPO in HDL particles.
6. HHCY were no effect of SOD and eNOS of HDL, the result need more research to confirm.
7. All of above, High Hcy may be an independent risk factor for coronary heart disease. HHCY reduce the RCT, anti-inflammatory and anti-antioxidant function of HDL to aggravate atherosclerosis. Reduce plasma homocysteine levels may improve the function of HDL, and reduce Hcy may become one of the targets for the treatment of atherosclerosis.
引文
[1]Overbaugh KJ:Acute coronary syndrome. Am J Nurs 2009,109(5):42-52, 53.
[2]Wong BW, Meredith A, Lin D, McManus BM:The biological role of inflammation in atherosclerosis. Can J Cardiol 2012,28(6):631-641.
[3]Erhardt LR, Leiter LA, and Hobbs FD:Lipid management in cardiovascular disease prevention guidelines:strategies and tactics for implementation. Atherosclerosis 2008,196(2):532-541.
[4]Schaefer JR:Lipid management for the prevention of cardiovascular disease. Curr Pharm Des 2011,17(9):852-860.
[5]Micha R, Mozaffarian D:Saturated fat and cardiometabolic risk factors, coronary heart disease, stroke, and diabetes:a fresh look at the evidence. Lipids 2010,45(10):893-905.
[6]Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation 2002, 106(25):3143-3421.
[7]Marma AK, Lloyd-Jones DM:Systematic examination of the updated Framingham heart study general cardiovascular risk profile. Circulation 2009, 120(5):384-390.
[8]Franceschini G:Epidemiologic evidence for high-density lipoprotein cholesterol as a risk factor for coronary artery disease. Am J Cardiol 2001, 88(12A):9N-13N.
[9]Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez-Sendon J, Mosca L, Tardif JC, Waters DD et al:Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med 2007, 357(21):2109-2122.
[10]Gomaraschi M, Baldassarre D, Amato M, Eligini S, Conca P, Sirtori CR, Franceschini Q Calabresi L:Normal vascular function despite low levels of high-density lipoprotein cholesterol in carriers of the Apolipoprotein A-I(Milano) mutant. Circulation 2007,116(19):2165-2172.
[11]Trigatti BL, Krieger M, Rigotti A:Influence of the HDL receptor SR-BI on lipoprotein metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol 2003,23(10):1732-1738.
[12]de la Llera MM, McGillicuddy FC, Hinkle CC, Byrne M, Joshi MR, Nguyen V, Tabita-Martinez J, Wolfe ML, Badellino K, Pruscino L et al:Inflammation modulates human HDL composition and function in vivo. Atherosclerosis 2012,222(2):390-394.
[13]Duffy D, Rader DJ:Update on strategies to increase HDL quantity and function. Nat Rev Cardiol 2009,6(7):455-463.
[14]Hazen S:HDL structure, function, therapeutics, and imaging. Arterioscler Thromb Vasc Biol 2010,30(2):138.
[15]Bashtovyy D, Jones MK, Anantharamaiah GM, and Segrest JP:Sequence conservation of Apolipoprotein A-I affords novel insights into HDL structure-function. J Lipid Res 2011,52(3):435-450.
[16]Marsche G, Saemann MD, and Heinemann A, Holzer M:Inflammation alters HDL composition and function:Implications for HDL-raising therapies. Pharmacol Ther 2013,137(3):341-351.
[17]Smith JD:Myeloperoxidase, inflammation, and dysfunctional high-density lipoprotein. J Clin Lipidol 2010,4(5):382-388.
[18]Yekini I, Hammoudi F, Martin-Nizard F, Yous S, Lebegue N, Berthelot P, Carato P:Antioxidant activity of benzoxazolinonic and benzothiazolinonic derivatives in the LDL oxidation model. Bioorg Med Chem 2009, 17(22):7823-7830.
[19]Navab M, Shechter I, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Fogelman AM:Structure and function of HDL mimetics. Arterioscler Thromb Vasc Biol 2010,30(2):164-168.
[20]Cesari M, Marzetti E, Laudisio A, Antonica L, Pahor M, Bernabei R, Zuccala G:Interaction of HDL cholesterol concentrations on the relationship between physical function and inflammation in community-dwelling older persons. Age Ageing 2010,39(1):74-80.
[21]Dodani S, Grice DG, Joshi S:Is HDL function as important as HDL quantity in the coronary artery disease risk assessment? J Clin Lipidol 2009, 3(2):70-77.
[22]Lesnik P, Chapman MJ:A new dimension in the vasculoprotective function of HDL:progenitor-mediated endothelium repair. Arterioscler Thromb Vase Biol 2006,26(5):965-967.
[23]Maiolino G, Pedon L, Cesari M, Frigo AC, Wolfert RL, Barisa M, Pagliani L, Rossitto G, and Seccia TM, Zanchetta M et al:Lipoprotein-associated phospholipase A2 activity predicts cardiovascular events in high risk coronary artery disease patients. PLoS One 2012,7(10):e48171.
[24]Robins SJ, Collins D, Nelson JJ, Bloomfield HE, Asztalos BF: Cardiovascular events with increased lipoprotein-associated phospholipase A(2) and low high-density lipoprotein-cholesterol:the Veterans Affairs HDL Intervention Trial. Arterioscler Thromb Vase Biol 2008,28(6):1172-1178.
[25]Antoniades C, Shirodaria C, Stefanadis C, Channon KM:Homocysteine lowering:any use in atherosclerosis? Hellenic J Candiol 2007, 48(5):249-251.
[26]Su J, Wang S, Hunag Y, Jinag Y:[A comparative study on pathogenic effects of homocysteine and cysteine on atherosclerosis]. Wei Sheng Yan Jiu 2009, 38(1):43-46.
[27]Guthikonda S, Haynes WG:Homocysteine:role and implications in atherosclerosis. Curr Atheroscler Rep 2006,8(2):100-106.
[28]Lin CP, Chen YH, Lin WT, Leu HB, Liu TZ, Huang SL, Chen JW:Direct effect of statins on homocysteine-induced endothelial adhesiveness:potential impact to human atherosclerosis. Eur J Clin Invest 2008,38(2):106-116.
[29]Della-Morte D, Beecham A, Rundek T, Slifer S, Boden-Albala B, McClendon MS, Blanton SH, Sacco RL:Genetic linkage of serum homocysteine in Dominican families:the Family Study of Stroke Risk and Carotid Atherosclerosis. Stroke 2010,41(7):1356-1362.
[30]Lever M, Slow S, George P M, et al. Betaine excretion correlates with plasma homocysteine when plasma lipids are elevate. Clinical biochemistry, 2012,45(1):154-156.
[31]Karalezli A, Parlak E S, Kanbay A, et al. Homocysteine and Serum-Lipid Levels in Pulmonary Embolism[J]. Clinical and Applied Thrombosis/Hemostasis,2011,17(6):E186-E189.
[32]Barter P J, Rye K A. Homocysteine and cardiovascular disease is HDL the link? Circulation research,2006,99(6):565-566.
[33]Perla-Kajan J, Jakubowski H. Paraoxonase 1 and homocysteine metabolism. Amino acids,2012,43(4):1405-1417.
[34]Yilmaz N. Relationship between paraoxonase and homocysteine:crossroads of oxidative diseases. Arch Med Sci,2012,8(1):138-153.
[35]Clifford A J, Chen K, Mc Wade L, et al. Gender and single nucleotide polymorphisms in MTHFR, BHMT, SPTLC1, CRBP2, CETP, and SCARB1 are significant predictors of plasma homocysteine normalized by RBC folate in healthy adults[J]. The Journal of nutrition,2012,142(9):1764-1771.
[36]Real J T, Martinez-Hervas S, Garcia-Garcia A B, et al. Association of C677T polymorphism in MTHFR gene, high homocysteine and low HDL cholesterol plasma values in heterozygous familial hypercholesterolemia. Journal of atherosclerosis and thrombosis,2009,16(6):815.
[37]Malinow MR, Bostom AG, Krauss RM:Homocyst(e)ine, diet, and cardiovascular diseases:a statement for healthcare professionals from the Nutrition Committee, American Heart Association. Circulation 1999, 99(1):178-182.
[38]Gensini G G. A more meaningful scoring system for determining the severity of coronary heart disease. Am J cardiol,1983,51:606..
[39]Blum A, Lupovitch S, Khazim K, Peleg A, Gumanovsky M, Yeganeh S, Jawabreh S:Homocysteine levels in patients with risk factors for atherosclerosis. Clin Cardiol 2001,24(6):463-466.
[40]Cheng TO:Homocysteine, B vitamins, and atherosclerosis. Circulation 1999, 99(3):459-460.
[41]Engeham SF, Haase A, and Langley-Evans SC:Supplementation of a maternal low-protein diet in rat pregnancy with folic acid ameliorates programming effects upon feeding behaviour in the absence of disturbances to the methionine-homocysteine cycle. Br J Nutr 2010,103(7):996-1007.
[42]Hou CT, Wu YH, Cheng CH, Huang PN, and Huang YC:Higher plasma homocysteine is associated with lower vitamin B6 status in critically ill surgical patients. Nutr Clin Pract 2012,27(5):695-700.
[43]Olszewski AJ, Szostak WB, Bialkowska M, Rudnicki S, and McCully KS: Reduction of plasma lipid and homocysteine levels by pyridoxine, folate, cobalamin, choline, riboflavin, and troxerutin in atherosclerosis. Atherosclerosis 1989,75(1):1-6.
[44]Malanovic N, Streith I, Wolinski H, Rechberger G, and Kohlwein SD, Tehlivets O:S-adenosyl-L-homocysteine hydrolase, key enzyme of methylation metabolism, regulates phosphatidylcholine synthesis and triacylglycerol homeostasis in yeast:implications for homocysteine as a risk factor of atherosclerosis. J Biol Chem 2008,283(35):23989-23999.
[45]Held C, Sumner G, Sheridan P, McQueen M, Smith S, Dagenais G, Yusuf S, Lonn E:Correlations between plasma homocysteine and folate concentrations and carotid atherosclerosis in high-risk individuals:baseline data from the Homocysteine and Atherosclerosis Reduction Trial (HART). Vasc Med 2008,13(4):245-253.
[46]All AK, Bobadilla RV:Preventing restenosis in CAD patients. Folate therapy reduces homocysteine--and risk. Adv Nurse Pract 2004,12(3):79-82.
[47]Cacciapuoti F:Lowering homocysteine levels with folic acid and B-vitamins do not reduce early atherosclerosis, but could interfere with cognitive decline and Alzheimer's disease. J Thromb Thrombolysis 2012.
[48]Holmes MV, Newcombe P, Hubacek JA, Sofat R, Ricketts SL, Cooper J, Breteler MM, Bautista LE, and Sharma P, Whittaker JC et al:Effect modification by population dietary folate on the association between MTHFR genotype, homocysteine, and stroke risk:a meta-analysis of genetic studies and randomised trials. Lancet 2011,378(9791):584-594.
[49]Han IB, Kim OJ, Ahn JY, Oh D, Hong SP, Huh R, Chung SS, Kim NK: Association of methylenetetrahydrofolate reductase (MTHFR 677C＞T and 1298A>C) polymorphisms and haplotypes with silent brain infarction and homocysteine levels in a Korean population. Yonsei Med J 2010, 51(2):253-260.
[50]Klai S, Fekih-Mrissa N, El HS, Kaabechi N, Nsiri B, Rachdi R, Gritli N: Association of MTHFR A1298C polymorphism (but not of MTHFR C677T) with elevated homocysteine levels and placental vasculopathies. Blood Coagul Fibrinolysis 2011,22(5):374-378.
[51]Hanta I, Soydas Y, Karatasli M, Koseoglu Z, Satar S, Hasturk S:Plasma homocysteine level and 677C-->T mutation on the MTHFR gene in patients with venous thromboembolism. Bratisl Lek Listy 2010,111(2):70-73.
[52]Murakami H, Iemitsu M, Sanada K, Gando Y, Ohmori Y, Kawakami R, Sasaki S, Tabata I, Miyachi M:Associations among objectively measured physical activity, fasting plasma homocysteine concentration, and MTHFR C677T genotype. Eur J Appl Physiol 2011,111(12):2997-3005.
[53]Ignashkova TI, Mesitov MV, Rybakov AS, Moskovtsev AA, Sokolovskaia AA, Kubatiev AA:[Deposition of von Willebrand factor in human endothelial cells HUVEC in the endoplasmic reticulum stress induced by an excess of homocysteine in vitro]. Patol Fiziol Eksp Ter 2012(3):81-86.
[54]Da CM, Da CA, Ferreira AG, Machado FR, Schmitz F, Lima DD, Delwing D, Mussulini BH, Wofchuk S, Netto CA et al:Physical exercise reverses glutamate uptake and oxidative stress effects of chronic homocysteine administration in the rat. Int J Dev Neurosci 2012,30(2):69-74.
[55]Caylak E, Aytekin M, Halifeoglu I:Antioxidant effects of methionine, alpha-lipoic acid, N-acetylcysteine and homocysteine on lead-induced oxidative stress to erythrocytes in rats. Exp Toxicol Pathol 2008, 60(4-5):289-294.
[56]Lin CP, Chen YH, Leu HB, Lin SJ, Chen YL, Huang SL, Chen JW: Anti-inflammatory strategies for homocysteine-related cardiovascular disease. Front Biosci 2009,14:3836-3845.
[57]Mansouri Z, Sabetkasaei M, Moradi F, and Masoudnia F, Ataie A:Curcumin has neuroprotection effect on homocysteine rat model of Parkinson. J Mol Neurosci 2012,47(2):234-242.
[58]Hotston M, Jeremy JY, Bloor J, Greaves NS, Persad R, and Angelini G, Shukla N:Homocysteine and copper interact to promote type 5 phosphodiesterase expression in rabbit cavernosal smooth muscle cells. Asian J Androl 2008,10(6):905-913.
[59]Javadzadeh A, Ghorbanihaghjo A, Bahreini E, Rashtchizadeh N, Argani H, Alizadeh S:Serum paraoxonase phenotype distribution in exudative age-related macular degeneration and its relationship to homocysteine and oxidized low-density lipoprotein. Retina 2012,32(4):658-666.
[60]Oravec S, Dostal E, Dukat A, Gavornik P, and Kucera M, Gruber K:HDL subfractions analysis:a new laboratory diagnostic assay for patients with cardiovascular diseases and dyslipoproteinemia. Neuro Endocrmol Lett 2011, 32(4):502-509.
[61]Kalofoutis A, Papapanagiotou A, Tzivras M:Clinical significance of plasma HDL subfractions (HDL2, HDL3) in patients with peripheral arterial disease (PAD) in the Greek population. Clin Biochem 1999,32(2):149-152.
[62]Filippatos TD, Liberopoulos EN, Kostapanos M, Gazi IF, Papavasiliou EC, Kiortsis DN, Tselepis AD, Elisaf MS:The effects of orlistat and fenofibrate, alone or in combination, on high-density lipoprotein subfractions and pre-betal-HDL levels in obese patients with metabolic syndrome. Diabetes Obes Metab 2008,10(6):476-483.
[63]Moffatt RJ, Biggerstaff KD, and Stamford BA:Effects of the transdermal nicotine patch on normalization of HDL-C and its subfractions. Prev Med 2000,31 (2 Pt 1):148-152.
[64]Harangi M, Seres I, Harangi J, Paragh G:Benefits and difficulties in measuring HDL subfractions and human paraoxonase-1 activity during statin treatment. Cardiovasc Drugs Ther 2009,23(6):501-510.
[65]Guo ZG, Li C, Zhong JK, Tu Y, Xie D:Laboratory investigation of dysfunctional HDL. Chem Phys Lipids 2012,165(1):32-37.
[66]Badeau RM, Metso J, Kovanen PT, Lee-Rueckert M, and Tikkanen MJ, Jauhiainen M:The impacts of gender and serum estradiol levels on HDL-mediated reverse cholesterol transport. Eur J Clin Invest 2012.
[67]Zhong JK, Guo ZG, Li C, Wang ZK, Lai WY, and Tu Y:Probucol alleviates atherosclerosis and improves high density lipoprotein function. Lipids Health Dis 2011,10:210.
[68]Rocco AG, Sensi C, Gianazza E, Calabresi L, Franceschini G, Sirtori CR, Eberini I:Structural and dynamic features of Apolipoprotein A-I cysteine mutants, Milano and Paris, in synthetic HDL. J Mol Graph Model 2010.
[69]Shah PK:Atherosclerosis:targeting endogenous Apo A-I--a new approach for raising HDL. Nat Rev Cardiol 2011,8(4):187-188.
[70]Speidl WS, Cimmino G, Ibanez B, Elmariah S, Hutter R, Garcia MJ, Fuster V, Goldman ME, Badimon JJ:Recombinant Apolipoprotein A-I Milano rapidly reverses aortic valve stenosis and decreases leaflet inflammation in an experimental rabbit model. Eur Heart J 2010,31(16):2049-2057.
[71]Bankston TE, Carta G:Apolipoprotein A-I(Milano) anion exchange chromatography:Self association and adsorption equilibrium. Biotechnol J 2010,5(10):1028-1039.
[72]Alexander ET, Tanaka M, Kono M, Saito H, Rader DJ, Phillips MC: Structural and functional consequences of the Milano mutation (R173C) in human Apolipoprotein A-I. J Lipid Res 2009,50(7):1409-1419.
[73]Roshan B, Ganda OP, Desilva R, Ganim RB, Ward E, Haessler SD, Polisecki EY, Asztalos BF, Schaefer EJ:Homozygous lecithin:cholesterol acyltransferase (LCAT) deficiency due to a new loss of function mutation and review of the literature. J Clin Lipidol 2011,5(6):493-499.
[74]Weers PM, Patel AB, Wan LC, Guigard E, Kay CM, Hafiane A, McPherson R, Marcel YL, Kiss RS:Novel N-terminal mutation of human Apolipoprotein A-I reduces self-association and impairs LCAT activation. J Lipid Res 2011,52(1):35-44.
[75]Hossain MA, Tsujita M, Akita N, Kobayashi F, Yokoyama S:Cholesterol homeostasis in ABCA1/LCAT double-deficient mouse. Biochim Biophys Acta 2009,1791(12):1197-1205.
[76]Rajpal JS, Mapellentz J, Mancera AD, Reed RC, Kim Y, Chavers BM: Familial LCAT deficiency in a child with nephrotic syndrome. Clin Nephrol 2013.
[77]von Eckardstein A:Mulling over the odds of CETP inhibition. Eur Heart J 2010,31(4):390-393.
[78]Charles MA, Kane JP:New molecular insights into CETP structure and function:a review. J Lipid Res 2012,53(8):1451-1458.
[79]Wang Y, Berbee JF, Stroes ES, Smit JW, Havekes LM, Romijn JA, Rensen PC:CETP expression reverses the reconstituted HDL-induced increase in VLDL.J Lipid Res 2011,52(8):1533-1541.
[80]Wang Y, Snel M, Jonker JT, Hammer S, Lamb HJ, de Roos A,Meinders AE, Pijl H, Romijn JA, Smit JW et al:Prolonged caloric restriction in obese patients with type 2 diabetes mellitus decreases plasma CETP and increases Apolipoprotein AI levels without improving the cholesterol efflux properties of HDL. Diabetes Care 2011,34(12):2576-2580.
[81]Davidson MH:Update on CETP inhibition. J Clin Lipidol 2010, 4(5):394-398.
[82]Bochem AE, Kuivenhoven JA, Stroes ES:The promise of Cholesteryl Ester Transfer Protein (CETP) inhibition in the treatment of cardiovascular disease. Curr Pharm Des 2013.
[83]Gugliucci A, Kinugasa E, Kotani K, Caccavello R, Kimura S:Serum paraoxonase 1 (PON1) lactonase activity is lower in end-stage renal disease patients than in healthy control subjects and increases after hemodialysis. Clin Chem Lab Med 2011,49(1):61-67.
[84]Duffy D, Rader DJ:Update on strategies to increase HDL quantity and function. Nat Rev Cardiol 2009,6(7):455-463.
[85]Hazen S:HDL structure, function, therapeutics, and imaging. Arterioscler Thromb Vasc Biol 2010,30(2):138.
[86]Hausenloy DJ, Yellon DM:Targeting residual cardiovascular risk:raising high-density lipoprotein cholesterol levels. Heart 2008,94(6):706-714.
[87]Gabay C, Kushner I:Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999,340(6):448-454.
[88]Riedmaier S, Klein K, Winter S, Hofmann U, Schwab M, Zanger UM: Paraoxonase (PON1 and PON3) Polymorphisms:Impact on Liver Expression and Atorvastatin-Lactone Hydrolysis. Front Pharmacol 2011, 2:41.
[89]Draganov D, Teiber J, Watson C, Bisgaier C, Nemzek J, Remick D, Standiford T, La Du B:PON1 and oxidative stress in human sepsis and an animal model of sepsis. Adv Exp Med Biol 2010,660:89-97.
[90]Mehdi MM, Rizvi SI:Human plasma paraoxonase 1 (PON1) arylesterase activity during aging:correlation with susceptibility of LDL oxidation. Arch Med Res 2012,43(6):438-443.
[91]Hine D, Mackness B, Mackness M:Coincubation of PON1, APO A1, and LCAT increases the time HDL is able to prevent LDL oxidation. lubmb Life 2012,64(2):157-161.
[92]Taylor LJ, Porter JM:Elevated plasma homocysteine as a risk factor for atherosclerosis. Semin Vasc Surg 1993,6(1):36-45.
[93]Koubaa N, Nakbi A, Hammami S, Attia N, Mehri S, Ben HK, Ben FM, Miled A, Hammami M:Association of homocysteine thiolactonase activity and PON1 polymorphisms with the severity of acute coronary syndrome. Clin Biochem 2009,42(9):771-776.
[94]Macharia M, Hassan MS, Blackhurst D, Erasmus RT, Matsha TE:The growing importance of PON1 in cardiovascular health:a review. J Cardiovasc Med (Hagerstown) 2012,13(7):443-453.
[95]Karakas M, Koenig W:Myeloperoxidase production by macrophage and risk of atherosclerosis. Curr Atheroscler Rep 2012,14(3):277-283.
[96]Nicholls SJ, Zheng L, Hazen SL:Formation of dysfunctional high-density lipoprotein by myeloperoxidase. Trends Cardiovasc Med 2005, 15(6):212-219.
[97]Tang WH, Wu Y, Nicholls SJ, Hazen SL:Plasma myeloperoxidase predicts incident cardiovascular risks in stable patients undergoing medical management for coronary artery disease. Clin Chem 2011,57(1):33-39.
[98]Samsamshariat SZ, Basati G, Movahedian A, Pourfarzam M, Sarrafzadegan N:Elevated plasma myeloperoxidase levels in relation to circulating inflammatory markers in coronary artery disease. Biomark Med 2011, 5(3):377-385.
[99]Zheng L, Nukuna B, Brennan ML, Sun M, Goormastic M, Settle M, Schmitt D, Fu X, Thomson L, Fox PL et al:Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease. J Clin Invest 2004,114(4):529-541.
[100]Ndrepepa G, Braun S, Mehilli J, von Beckerath N, Schomig A, Kastrati A: Myeloperoxidase level in patients with stable coronary artery disease and acute coronary syndromes. Eur J Clin Invest 2008,38(2):90-96.
[101]Wong CK, White HD:Effects of dietary factors on lipoprotein-associated phospholipase A(2) (Lp-PLA (2)). Curr Atheroscler Rep 2011, 13(6):461-466.
[102]Chae JS, Kim OY, Paik JK, Kang R, Seo WJ, Jeong TS, Sweeney G, Lee SH, Lee JH:Association of Lp-PLA(2) activity and LDL size with interleukin-6, an inflammatory cytokine and oxidized LDL, a marker of oxidative stress, in women with metabolic syndrome. Atherosclerosis 2011,218(2):499-506.
[103]Packard CJ, O'Reilly DS, Caslake MJ, McMahon AD, Ford I, Cooney J, Macphee CH, Suckling KE, Krishna M, Wilkinson FE et al: Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. West of Scotland Coronary Prevention Study Group. N Engl J Med 2000,343(16):1148-1155.
[104]Brilakis ES, Khera A, Saeed B, Banerjee S, McGuire DK, Murphy SA, de Lemos JA:Association of lipoprotein-associated phospholipase A2 mass and activity with coronary and aortic atherosclerosis:findings from the Dallas Heart Study. Clin Chem 2008,54(12):1975-1981.
[105]Lahdesmaki K, Oorni K, Alanne-Kinnunen M, Jauhiainen M, Hurt-Camejo E, Kovanen PT:Acidity and lipolysis by group V secreted phospholipase A(2) strongly increase the binding of ApoB-100-containing lipoproteins to human aortic proteoglycans. Biochim Biophys Acta 2012,1821(2):257-267.
[106]Gaubatz JW, Gillard BK, Massey JB, Hoogeveen RC, Huang M, Lloyd EE, Raya JL, Yang CY, Pownall HJ:Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A(2). J Lipid Res 2007,48(2):348-357.
[107]Silva IT, Mello AP, Damasceno NR:Antioxidant and inflammatory aspects of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)):a review. Lipids Health Dis 2011,10:170.
[108]Rallidis LS, Tellis CC, Lekakis J, Rizos I, Varounis C, Charalampopoulos A, Zolindaki M, Dagres N, Anastasiou-Nana M, Tselepis AD: Lipoprotein-associated phospholipase A(2) bound on high-density lipoprotein is associated with lower risk for cardiac death in stable coronary artery disease patients:a 3-year follow-up. J Am Coll Cardiol 2012, 60(20):2053-2060.
[109]Volchegorskii IA, Malinovskaya NV, Shumelyova OV, Shiemyakov SE: Dynamics of LPO products and oxidative modification of proteins in human brain during postnatal development. Bull Exp Biol Med 2007, 144(2):192-199.
[110]Solin AV, Lyashev YD:Effect of opioid peptides on the content of LPO products and antioxidant enzyme activity in the liver of rats after restraint stress. Bull Exp Biol Med 2012,153(6):827-829.
[111]Decorde K, Ventura E, Lacan D, Ramos J, Cristol JP, Rouanet JM:An SOD rich melon extract Extramel prevents aortic lipids and liver steatosis in diet-induced model of atherosclerosis. Nutr Metab Cardiovasc Dis 2010, 20(5):301-307.
[112]Zawadzka-Bartczak E:Activities of red blood cell anti-oxidative enzymes (SOD, GPx) and total anti-oxidative capacity of serum (TAS) in men with coronary atherosclerosis and in healthy pilots. Med Sci Monit 2005, 11(9):R440-R444.
[113]Takaya T, Hirata K, Yamashita T, Shinohara M, Sasaki N, Inoue N, Yada T, Goto M, Fukatsu A, Hayashi T et al:A specific role for eNOS-derived reactive oxygen species in atherosclerosis progression. Arterioscler Thromb Vasc Biol 2007,27(7):1632-1637.
[114]Ota H, Eto M, Ogawa S, Iijima K, Akishita M, Ouchi Y:SIR11/eNOS axis as a potential target against vascular senescence, dysfunction and atherosclerosis. J Atheroscler Thromb 2010,17(5):431-435.