高效液相色谱—串联质谱多反应监测鉴定人精浆脂质成分
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摘要
目的鉴定男性精浆中脂质成分,为其在生殖领域中的研究和应用提供参考。方法采用高效液相色谱—串联质谱(HPLC-MS/MS)多反应监测技术,对人精浆中脂质类化合物成分及含量进行分析。结果共鉴定出24个脂质大类,包含273种脂质种类。按化学结构可分为5类:甾醇类,包括游离胆固醇(Cho)、胆固醇酯(CE);甘油酯类,包括三酰甘油(TAG)、二酰甘油(DAG);磷脂酰胆碱类,包括卵磷脂(PC)、磷脂酰乙醇胺(PE)、磷脂酰甘油(PG)、磷脂酰肌醇(PI)、磷脂酰丝氨酸(PS)、磷脂酸(PA);溶血磷脂类,包括溶血磷脂二酸(LBPA)、溶血磷脂酰胆碱(LPC)、溶血磷脂酰乙醇胺(LPE)、溶血磷脂酸(LPA)、溶血磷脂酰肌醇(LPI)、溶血磷脂酰丝氨酸(LPS);鞘脂类,包括鞘磷脂(SM)、神经酰胺(Cer)、葡糖神经酰胺(Glu Cer)、乳糖神经酰胺(Lac Cer)、神经节苷脂(GM3)、红细胞糖鞘脂(Gb3)、1-磷酸鞘氨(S1P)、鞘氨醇(Sph)。在这24个脂质大类中,相对含量最高的前3类分别是Cho、SM和PC,包含种类最多的前3类分别是PC、PE和TAG。结论应用脂质组学分析方法显示男性精浆脂质,为今后分析不同病理状态下精浆内各种脂质代谢物的变化、了解脂质代谢的特征提供数据基础,为将来通过HPLC-MS/MS脂质组学技术发掘可预测精子质量的敏感、特异的脂质标志物提供新技术手段。
Objective To identify the lipid composition in male seminal plasma, thus providing reference for the study and application of lipidomics in the field of male reproduction. Methods High performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) was used to analyze the composition and content of lipids in human seminal plasma. Results A total of 24 lipid classes including 273 species were identified. According to the chemical structure, these 24 classes could be divided into 5 categories: sterols[including free cholesterols(Cho), cholesterol ester(CE)]; glycerides [including triacylglycerols(TAG), diacylglycerols(DAG)]; phosphatidylcholines [including phosphatidylcholines(PC), phosphatidylethanolamines(PE), phosphatidylglycerols(PG), phosphatidylinositols(PI), phophatidylserines(PS), phosphatidic acids(PA)]; lysophospholipids [including lyso-bisphosphatidic acids(LBPA), lyso-PC(LPC), lyso-PE(LPE),lyso-PA(LPA), lyso-PI(LPI), lyso-PS(LPS)] and sphingolipids [including sphingomyelins(SM), ceramides(Cer), glucosylceramides(Glu Cer), lactosylceramides(Lac Cer), monosialo-dihexosyl gangliosides(GM3), Globotriaosylceramide(Gb3), sphingosine-1-phosphates(S1 P), sphingosines(Sph)]. Of these 24 lipid classes, the top 3 with a relative content were Cho, SM and PC; the top 3 with a species number were PC, PE and TAG. Conclusion Through systematic and sensitive analytical methods, we displayed lipidomics of male seminal plasma, thus providing a data basis for analyzing the changes of lipid metabolites of male seminal plasma in different pathological conditions and comprehending the characteristics of lipid metabolism and, meanwhile, providing new means for exploring lipid markers that can sensitively and specially predict the semen quality with the technology of HPLC-MS/MS.
Objective To identify the lipid composition in male seminal plasma, thus providing reference for the study and application of lipidomics in the field of male reproduction. Methods High performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) was used to analyze the composition and content of lipids in human seminal plasma. Results A total of 24 lipid classes including 273 species were identified. According to the chemical structure, these 24 classes could be divided into 5 categories: sterols[including free cholesterols(Cho), cholesterol ester(CE)]; glycerides [including triacylglycerols(TAG), diacylglycerols(DAG)]; phosphatidylcholines [including phosphatidylcholines(PC), phosphatidylethanolamines(PE), phosphatidylglycerols(PG), phosphatidylinositols(PI), phophatidylserines(PS), phosphatidic acids(PA)]; lysophospholipids [including lyso-bisphosphatidic acids(LBPA), lyso-PC(LPC), lyso-PE(LPE),lyso-PA(LPA), lyso-PI(LPI), lyso-PS(LPS)] and sphingolipids [including sphingomyelins(SM), ceramides(Cer), glucosylceramides(Glu Cer), lactosylceramides(Lac Cer), monosialo-dihexosyl gangliosides(GM3), Globotriaosylceramide(Gb3), sphingosine-1-phosphates(S1 P), sphingosines(Sph)]. Of these 24 lipid classes, the top 3 with a relative content were Cho, SM and PC; the top 3 with a species number were PC, PE and TAG. Conclusion Through systematic and sensitive analytical methods, we displayed lipidomics of male seminal plasma, thus providing a data basis for analyzing the changes of lipid metabolites of male seminal plasma in different pathological conditions and comprehending the characteristics of lipid metabolism and, meanwhile, providing new means for exploring lipid markers that can sensitively and specially predict the semen quality with the technology of HPLC-MS/MS.
引文
[1]江凡,邹建华,张修发.生殖激素测定在男性不育病因学诊断中的临床价值[J].临床和实验医学杂志,2014,13(16):1361—1363
[2]杜金龙,屈爱春.实验室精液检验与男性不育原因探讨[J].中国医学创新,2010,7(4):143—144
[3]张红烨,陆金春,冯瑞祥.24种精浆生化指标与精液常规参数的相关性研究[J].中华男科学杂志,2015,21(12):1087—1092
[4]Vignon F,Clavert A,Cranz C,et al.Alterations in the lipid composition of seminal plasma in patients with a chronic infection of the urogenital tract[J].Urol Int,1993,50(1):36—38
[5]Gomathi C,Balasubramanian K,Bhanu NV,et al.Effect of chronic alcoholism on semen-studies on lipid profiles[J].Int J Androl,1993,16(3):175—181
[6]Zura ZI,Samardzija M,Vince S,et al.Differences in seminal plasma and spermatozoa antioxidative systems and seminal plasma lipid and protein levels among boar breeds and hybrid genetic traits[J].Anim Reprod Sci,2016,170:75—82
[7]Argov-Argaman N,Mahgrefthe K,Zeron Y,et al.Variation in lipid profiles within semen compartments-the bovine model of aging[J].Theriogenology,2013,80(7):712—721
[8]张娟红,王荣,王立娟,等.急进高原对甲氧氯普胺蛋白结合率及药代动力学参数的影响[J].兰州大学学报:医学版,2017,43(3):13—17
[9]Schiller J,Suss R,Arnhold J,et al.Matrix-assisted laser desorption and ionization time-of-flight(MALDI-TOF)mass spectrometry in lipid and phospholipid research[J].Prog Lipid Res,2004,43(5):449—488
[10]Wenk MR.Lipidomics:new tools and applications[J].Cell,2010,143(6):888—895
[11]Christinat N,Morin-Rivron D,Masoodi M.Highthroughput quantitative lipidomics analysis of nonesterified fatty acids in human plasma[J].J Proteome Res,2016,15(7):2228—2235
[12]Shevchenko A,Simons K.Lipidomics:coming to grips with lipid diversity[J].Nat Rev Mol Cell Biol,2010,11(8):593—598
[13]倪晓佳,王占璋,卢浩扬,等.Hplc-Ms/Ms法同时测定人血清中利培酮与帕利哌酮及奥氮平和喹硫平的浓度[J].中国临床药理学杂志,2018,34(1):53—56
[14]吴健美,刘培培,樊颖锋.Hplc-Ms/Ms法同时测定尿液中的常见毒品及其代谢物和常见药物[J].警察技术,2018,25(1):79—82
[15]励炯,金朦娜,李玮,等.实心核颗粒色谱分离-HplcMs/Ms法同时测定性保健品中非法添加15种壮阳类化学成分[J].中草药,2018,49(3):604—609
[16]Lam SM,Chua GH,Li XJ,et al.Biological relevance of fatty acyl heterogeneity to the neural membrane dynamics of rhesus macaques during normative aging[J].Oncotarget,2016,7(35):55970—55989
[17]Courant F,Antignac JP,Monteau F,et al.Metabolomics as a potential new approach for investigating human reproductive disorders[J].J Proteome Res,2013,12(6):2914—2920
[18]Han X,Gross RW.Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry:a bridge to lipidomics[J].J Lipid Res,2003,44(6):1071—1079
[19]谭淑瑜,朴胜华.脂质组学在中药调脂研究中的应用进展[J].亚太传统医药,2017,13(9):60—63
[20]洪赟,厉有名,虞朝晖.脂质组学技术在非酒精性脂肪性肝病中的研究进展[J].国际消化病杂志,2013,33(6):384—385
[21]陈凯云,于雪峰,刘贞,等.基于代谢组学技术对中西药干预股骨头坏死后甘油三脂代谢分析[J].中华中医药杂志,2016,31(11):4452—4455
[22]靳永文,张帆,石阿茜,等.美罗培南对丙戊酸钠血药浓度和脑组织分布的影响[J].兰州大学学报:医学版,2017,43(3):18—22
[23]Rolim AE,Henrique-Araujo R,Ferraz EG,et al.Lipidomics in the study of lipid metabolism:current perspectives in the omic sciences[J].Gene,2015,554(2):131—139
[24]Ivanova PT,Milne SB,Myers DS,et al.Lipidomics:a mass spectrometry based systems level analysis of cellular lipids[J].Curr Opin Chem Biol,2009,13(5—6):526—531
[25]Ferreira CR,Saraiva SAm,Catharino RR,et al.Single embryo and oocyte lipid fingerprinting by mass spectrometry[J].J Lipid Res,2010,51(5):1218—1227
[26]Cataldi T,Cordeiro FB,Costa LV,et al.Lipid profil young poor ovarian responders versus normal responders[J].Hum Fertil(Camb),2013,16(4):269—277
[27]Dennis EA.Lipidomics joins the omics evolution[J].Proc Natl Acad Sci USA,2009,106(7):2089—2090
[28]Li M,Feng B,Liang Y,et al.Lipid profiling of human plasma from peritoneal dialysis patients using an improved 2D(NP/RP)LC-QTo F MS Method[J].Anal Bioanal Chem,2013,405(21):6629—6638
[29]Li M,Tong X,LüP,et al.A not-stop-flow online normal-/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry method for comprehensive lipid profiling of human plasma from atherosclerosis patients[J].J Chromatogr A,2014,13(72):110—119
[30]Tang W,Li M,Lu XH,et al.Phospholipids profiling and outcome of peritoneal dialysis patients[J].Biomarkers,2014,19(6):505—508
[31]Yang L,Cui X,Zhang N,et al.Comprehensive lipid profiling of plasma in patients with benign breast tumor and breast cancer reveals novel biomarkers[J].Anal Bioanal Chem,2015,407(17):5065—5077
[32]朱超,胡坪,梁琼麟,等.脂组学及其研究进展[J].分析化学,2009,37(9):1390—1396
[33]Byskov AG,Andersen CY,Nordholm L,et al.Chemical structure of sterols that activate oocyte meiosis[J].Nature,1995,374(6522):559—562
[34]Stival C,Puga MLC,Paudel B,et al.Sperm capacitation and acrosome reaction in mammalian sperm[J].Adv Anat Embryol Cell Biol,2016,220:93—106
[35]Keber R,Rozman D,Horvat S.Sterols in spermatogenesis and sperm maturation[J].J Lipid Res,2013,54(1):20—33
[36]Cohen R,Mukai C,Travis AJ.Lipid regulation of acrosome exocytosis[J].Adv Anat Embryol Cell Biol,2016,220:107—127
[37]Lessig J,Glander HJ,Schiller J,et al.Destabilization of the acrosome results in release of phospholipase A2from human spermatozoa and subsequent formation of lysophospholipids[J].Andrologia,2006,38(2):69—75
[38]Fuchs B,Muller K,Paasch U,et al.Lysophospholipids:potential markers of diseases and infertility[J].Mini Rev Med Chem,2012,12(1):74—86
[39]Svetlichnyy V,Muller P,Pomorski TG,et al.Metabolic incorporation of unsaturated fatty acids into boar spermatozoa lipids and de novo formation of diacylglycerols[J].Chem Phys Lipids,2014,177:41—50
[40]Lopez CI,Pelletan LE,Suhaiman L,et al.Diacylglycerol stimulates acrosomal exocytosis by feeding into a PKC-and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate[J].Biochim Biophys Acta,2012,1821(9):1186—1199
[41]Petcoff DW,Holland WL,Stith BJ.Lipid levels in sperm,eggs,and during fertilization in xenopus laevis[J].J Lipid Res,2008,49(11):2365—2378
[42]Hajiahmadian M,Sarvi MK,Agh N,et al.Semen characteristics of rainbow trout(oncorhynchus mykiss)following diets containing different vegetable fatty acid levels[J].Reprod Domest Anim,2016,51(6):979—984
[43]Wang C,Huang X.Lipid metabolism and drosophila sperm development[J].Sci China Life Sci,2012,55(1):35—40
[44]Wood PL,Scoggin K,Ball BA,et al.Lipidomics of equine sperm and seminal plasma:identification of amphiphilic(o-acyl)-omega-hydroxy-fatty acids[J].Theriogenology,2016,86(5):1212—1221
[45]Lucio CF,Brito MM,Angrimani D,et al.Lipid composition of the canine sperm plasma membrane as markers of sperm motility[J].Reprod Domest Anim,2017,52(Suppl 2):208—213
[46]Kovac JR,Pastuszak AW,Lamb DJ.The use of genomics,proteomics,and metabolomics in identifying biomarkers of male infertility[J].Fertil Steril,2013,99(4):998—1007
[47]尹秋生,刘玉娥,韩玲霞.支原体感染精浆中脂质成分变化与男性不育症的关系[J].中国优生与遗传杂志,2002,10(3):111
[48]Gomathi C,Balasubramanian K,Bhanu NV,et al.Effect of chronic alcoholism on semen-studies on lipid profiles[J].Int J Androl,1993,16(3):175—181
[2]杜金龙,屈爱春.实验室精液检验与男性不育原因探讨[J].中国医学创新,2010,7(4):143—144
[3]张红烨,陆金春,冯瑞祥.24种精浆生化指标与精液常规参数的相关性研究[J].中华男科学杂志,2015,21(12):1087—1092
[4]Vignon F,Clavert A,Cranz C,et al.Alterations in the lipid composition of seminal plasma in patients with a chronic infection of the urogenital tract[J].Urol Int,1993,50(1):36—38
[5]Gomathi C,Balasubramanian K,Bhanu NV,et al.Effect of chronic alcoholism on semen-studies on lipid profiles[J].Int J Androl,1993,16(3):175—181
[6]Zura ZI,Samardzija M,Vince S,et al.Differences in seminal plasma and spermatozoa antioxidative systems and seminal plasma lipid and protein levels among boar breeds and hybrid genetic traits[J].Anim Reprod Sci,2016,170:75—82
[7]Argov-Argaman N,Mahgrefthe K,Zeron Y,et al.Variation in lipid profiles within semen compartments-the bovine model of aging[J].Theriogenology,2013,80(7):712—721
[8]张娟红,王荣,王立娟,等.急进高原对甲氧氯普胺蛋白结合率及药代动力学参数的影响[J].兰州大学学报:医学版,2017,43(3):13—17
[9]Schiller J,Suss R,Arnhold J,et al.Matrix-assisted laser desorption and ionization time-of-flight(MALDI-TOF)mass spectrometry in lipid and phospholipid research[J].Prog Lipid Res,2004,43(5):449—488
[10]Wenk MR.Lipidomics:new tools and applications[J].Cell,2010,143(6):888—895
[11]Christinat N,Morin-Rivron D,Masoodi M.Highthroughput quantitative lipidomics analysis of nonesterified fatty acids in human plasma[J].J Proteome Res,2016,15(7):2228—2235
[12]Shevchenko A,Simons K.Lipidomics:coming to grips with lipid diversity[J].Nat Rev Mol Cell Biol,2010,11(8):593—598
[13]倪晓佳,王占璋,卢浩扬,等.Hplc-Ms/Ms法同时测定人血清中利培酮与帕利哌酮及奥氮平和喹硫平的浓度[J].中国临床药理学杂志,2018,34(1):53—56
[14]吴健美,刘培培,樊颖锋.Hplc-Ms/Ms法同时测定尿液中的常见毒品及其代谢物和常见药物[J].警察技术,2018,25(1):79—82
[15]励炯,金朦娜,李玮,等.实心核颗粒色谱分离-HplcMs/Ms法同时测定性保健品中非法添加15种壮阳类化学成分[J].中草药,2018,49(3):604—609
[16]Lam SM,Chua GH,Li XJ,et al.Biological relevance of fatty acyl heterogeneity to the neural membrane dynamics of rhesus macaques during normative aging[J].Oncotarget,2016,7(35):55970—55989
[17]Courant F,Antignac JP,Monteau F,et al.Metabolomics as a potential new approach for investigating human reproductive disorders[J].J Proteome Res,2013,12(6):2914—2920
[18]Han X,Gross RW.Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry:a bridge to lipidomics[J].J Lipid Res,2003,44(6):1071—1079
[19]谭淑瑜,朴胜华.脂质组学在中药调脂研究中的应用进展[J].亚太传统医药,2017,13(9):60—63
[20]洪赟,厉有名,虞朝晖.脂质组学技术在非酒精性脂肪性肝病中的研究进展[J].国际消化病杂志,2013,33(6):384—385
[21]陈凯云,于雪峰,刘贞,等.基于代谢组学技术对中西药干预股骨头坏死后甘油三脂代谢分析[J].中华中医药杂志,2016,31(11):4452—4455
[22]靳永文,张帆,石阿茜,等.美罗培南对丙戊酸钠血药浓度和脑组织分布的影响[J].兰州大学学报:医学版,2017,43(3):18—22
[23]Rolim AE,Henrique-Araujo R,Ferraz EG,et al.Lipidomics in the study of lipid metabolism:current perspectives in the omic sciences[J].Gene,2015,554(2):131—139
[24]Ivanova PT,Milne SB,Myers DS,et al.Lipidomics:a mass spectrometry based systems level analysis of cellular lipids[J].Curr Opin Chem Biol,2009,13(5—6):526—531
[25]Ferreira CR,Saraiva SAm,Catharino RR,et al.Single embryo and oocyte lipid fingerprinting by mass spectrometry[J].J Lipid Res,2010,51(5):1218—1227
[26]Cataldi T,Cordeiro FB,Costa LV,et al.Lipid profil young poor ovarian responders versus normal responders[J].Hum Fertil(Camb),2013,16(4):269—277
[27]Dennis EA.Lipidomics joins the omics evolution[J].Proc Natl Acad Sci USA,2009,106(7):2089—2090
[28]Li M,Feng B,Liang Y,et al.Lipid profiling of human plasma from peritoneal dialysis patients using an improved 2D(NP/RP)LC-QTo F MS Method[J].Anal Bioanal Chem,2013,405(21):6629—6638
[29]Li M,Tong X,LüP,et al.A not-stop-flow online normal-/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry method for comprehensive lipid profiling of human plasma from atherosclerosis patients[J].J Chromatogr A,2014,13(72):110—119
[30]Tang W,Li M,Lu XH,et al.Phospholipids profiling and outcome of peritoneal dialysis patients[J].Biomarkers,2014,19(6):505—508
[31]Yang L,Cui X,Zhang N,et al.Comprehensive lipid profiling of plasma in patients with benign breast tumor and breast cancer reveals novel biomarkers[J].Anal Bioanal Chem,2015,407(17):5065—5077
[32]朱超,胡坪,梁琼麟,等.脂组学及其研究进展[J].分析化学,2009,37(9):1390—1396
[33]Byskov AG,Andersen CY,Nordholm L,et al.Chemical structure of sterols that activate oocyte meiosis[J].Nature,1995,374(6522):559—562
[34]Stival C,Puga MLC,Paudel B,et al.Sperm capacitation and acrosome reaction in mammalian sperm[J].Adv Anat Embryol Cell Biol,2016,220:93—106
[35]Keber R,Rozman D,Horvat S.Sterols in spermatogenesis and sperm maturation[J].J Lipid Res,2013,54(1):20—33
[36]Cohen R,Mukai C,Travis AJ.Lipid regulation of acrosome exocytosis[J].Adv Anat Embryol Cell Biol,2016,220:107—127
[37]Lessig J,Glander HJ,Schiller J,et al.Destabilization of the acrosome results in release of phospholipase A2from human spermatozoa and subsequent formation of lysophospholipids[J].Andrologia,2006,38(2):69—75
[38]Fuchs B,Muller K,Paasch U,et al.Lysophospholipids:potential markers of diseases and infertility[J].Mini Rev Med Chem,2012,12(1):74—86
[39]Svetlichnyy V,Muller P,Pomorski TG,et al.Metabolic incorporation of unsaturated fatty acids into boar spermatozoa lipids and de novo formation of diacylglycerols[J].Chem Phys Lipids,2014,177:41—50
[40]Lopez CI,Pelletan LE,Suhaiman L,et al.Diacylglycerol stimulates acrosomal exocytosis by feeding into a PKC-and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate[J].Biochim Biophys Acta,2012,1821(9):1186—1199
[41]Petcoff DW,Holland WL,Stith BJ.Lipid levels in sperm,eggs,and during fertilization in xenopus laevis[J].J Lipid Res,2008,49(11):2365—2378
[42]Hajiahmadian M,Sarvi MK,Agh N,et al.Semen characteristics of rainbow trout(oncorhynchus mykiss)following diets containing different vegetable fatty acid levels[J].Reprod Domest Anim,2016,51(6):979—984
[43]Wang C,Huang X.Lipid metabolism and drosophila sperm development[J].Sci China Life Sci,2012,55(1):35—40
[44]Wood PL,Scoggin K,Ball BA,et al.Lipidomics of equine sperm and seminal plasma:identification of amphiphilic(o-acyl)-omega-hydroxy-fatty acids[J].Theriogenology,2016,86(5):1212—1221
[45]Lucio CF,Brito MM,Angrimani D,et al.Lipid composition of the canine sperm plasma membrane as markers of sperm motility[J].Reprod Domest Anim,2017,52(Suppl 2):208—213
[46]Kovac JR,Pastuszak AW,Lamb DJ.The use of genomics,proteomics,and metabolomics in identifying biomarkers of male infertility[J].Fertil Steril,2013,99(4):998—1007
[47]尹秋生,刘玉娥,韩玲霞.支原体感染精浆中脂质成分变化与男性不育症的关系[J].中国优生与遗传杂志,2002,10(3):111
[48]Gomathi C,Balasubramanian K,Bhanu NV,et al.Effect of chronic alcoholism on semen-studies on lipid profiles[J].Int J Androl,1993,16(3):175—181