高效液相色谱法评价海洋生物中半胱亚磺酸脱羧酶活性
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摘要
[目的]建立磺基丙氨酸(CA)的高效液相色谱分析法,并以CA为底物,评价不同海洋生物组织中半胱亚磺酸脱羧酶(CSD)的活性。[方法]以Agilent Eclipse XDB-C_(18)为色谱柱,采用邻苯二甲醛(OPA)进行柱前衍生,检测波长为315 nm,对衍生化过程及色谱条件进行了优化。[结果]当CA∶OPA(物质的摩尔比)=1∶2、衍生时间2 min、流动相甲醇∶乙酸钠=65∶35(V/V)、流速1 m L/min时,CA具有最优的检测效果。在该条件下,CA浓度在10～500μg/m L具有良好的线性关系(R~2>0.99),平行样品的相对标准偏差为0.9%～8.6%,保留时间为1.4～1.5 min。不同海洋生物组织中CSD活性检测结果显示,在所选择的海洋贝类、头足类、鱼类中,贻贝、疣荔枝螺和大黄鱼均未检测到CSD活性;蛏子、血蛤、芝麻螺和鱿鱼中CSD活性较低。不同组织比较,海鲈鱼肝脏中CSD活性最高,达21.8 U/mg,而肉和鳃中则未检测到CSD活性。[结论]不同海洋生物体内牛磺酸的合成途径不尽相同。
[Objective] A method of high performance liquid chromatography( HPLC) was established and used to evaluate the activities of cysteinesulfinate decarboxylase( CSD) in different marine life by determining the cysteic acid( CA).[Method]The Agilent Eclipse XDB-C_(18) column was used for pre-column derivatization with o-phthalaldehyde( OPA) at a detection wavelength of 315 nm. The derivatization process and chromatographic conditions were optimized.[Result]The optimal condition for CA analysis was: CA ∶ OPA = 1 ∶ 2( n/n),derivatization time 2 min,the mobile phase methanol ∶ sodium acetate = 65 ∶ 35( V/V),and the flow rate of 1 m L/min. The relative standard deviations( RSD) of the parallel samples were 0.9%-8.6% and the retention time was 1.4-1.5 min. The linear range( R~2> 0.99) was in the range of 10-500 μg/m L. The results of CSD activity detection in different marine organisms showed that the CSD activity was not detected in mussel,rock whelk and large yellow croaker,but very low in razor clam,blood clams,furrowed planaxis and sleeve-fish. The activity of CSD in the liver of sea perch was the highest,reaching 21.8 U/mg,while it was not detected in meat and gill. [Conclusion]The synthesis pathways of taurine in different marine organisms are different.
[Objective] A method of high performance liquid chromatography( HPLC) was established and used to evaluate the activities of cysteinesulfinate decarboxylase( CSD) in different marine life by determining the cysteic acid( CA).[Method]The Agilent Eclipse XDB-C_(18) column was used for pre-column derivatization with o-phthalaldehyde( OPA) at a detection wavelength of 315 nm. The derivatization process and chromatographic conditions were optimized.[Result]The optimal condition for CA analysis was: CA ∶ OPA = 1 ∶ 2( n/n),derivatization time 2 min,the mobile phase methanol ∶ sodium acetate = 65 ∶ 35( V/V),and the flow rate of 1 m L/min. The relative standard deviations( RSD) of the parallel samples were 0.9%-8.6% and the retention time was 1.4-1.5 min. The linear range( R~2> 0.99) was in the range of 10-500 μg/m L. The results of CSD activity detection in different marine organisms showed that the CSD activity was not detected in mussel,rock whelk and large yellow croaker,but very low in razor clam,blood clams,furrowed planaxis and sleeve-fish. The activity of CSD in the liver of sea perch was the highest,reaching 21.8 U/mg,while it was not detected in meat and gill. [Conclusion]The synthesis pathways of taurine in different marine organisms are different.
引文
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[11]GOTO T,MATSUMOTO T,TAKAGI S.Distribution of the hepatic cysteamine dioxygenase activites in fish[J].Fisheries science,2001,67(6):1187-1189.
[12]TAPPAZ M,ALMARGHINI K,LEGAY F,et al.Taurine biosynthesis enzyme cysteine sulfinate decarboxylase(CSD)from brain:The long and tricky trail to identification[J].Neurochemical research,1992,17(9):849-859.
[13]GOTO T,MATSUMOTO T,MURAKAMI S,et al.Conversion of cysteate into taurine in liver of fish[J].Fisheries science,2003,69(1):216-218.
[14]UBUKA T,OKADA A,NAKAMURA H.Production of hypotaurine from L-cysteinesulfinate by rat liver mitochondria[J].Amino acids,2008,35(1):53-58.
[15]叶思平,陈悦娇,陈海光,等.高效液相色谱测定益智仁中的牛磺酸含量[J].食品科学,2013,34(10):204-206.
[16]陈申如,胡阳,倪辉,等.高效液相色谱法测定牡蛎中牛磺酸含量[J].中国食品学报,2013,13(2):193-198.
[17]柳小英,毛丽沙,鲍敏洁,等.高效液相色谱法测定海洋生物中牛磺酸的含量[J].安徽农业科学,2017,45(31):1-3,11.
[2]MIYAZAKI T,MATSUZAKI Y.Taurine and liver diseases:A focus on the heterogeneous protective properties of taurine[J].Amino acids,2014,46(1):101-110.
[3]PINTO W,RNNESTAD I,JORDAL A E O,et al.Cloning,tissue and ontogenetic expression of the taurine transporter in the flatfish Senegalese sole(Solea senegalensis)[J].Amino acids,2012,42(4):1317-1327.
[4]HIGUCHI M,CELINO F T,TAMAI A,et al.The synthesis and role of taurine in the Japanese eel testis[J].Amino acids,2012,43(2):773-781.
[5]MATSUNARI H,HASHIMOTO H,IWASAKI T,et al.Effect of feeding rotifers enriched with taurine on the growth and survival of larval amberjack Seriola dumerili[J].Fisheries science,2013,79(5):815-821.
[6]KIM C,CHA Y N.Taurine chloramine produced from taurine under inflammation provides anti-inflammatory and cytoprotective effects[J].Amino acids,2014,46(1):89-100.
[7]费东亮,王宏军.牛磺酸对染镉鸡脾淋巴细胞膜氧化损伤的保护作用[J].畜牧与兽医,2014,46(8):65-67.
[8]曾德寿,高振华,赵京辉,等.牛磺酸对肉仔鸡生产性能、免疫器官发育及抗氧化能力的影响[J].中国兽医学报,2009,29(6):774-778.
[9]RAK K,VOLKER J,JURGENS L,et al.Neurotrophic effects of taurine on spiral ganglion neurons in vitro[J].Neuroreport,2014,25(16):1250-1254.
[10]FUNKE S,AZIMI D,WOLTERS D,et al.Longitudinal analysis of taurine induced effects on the tear proteome of contact lens wearers and dry eye patients using a RP-RP-Capillary-HPLC-MALDI TOF/TOF MS approach[J].Journal of proteomics,2012,75(11):3177-3190.
[11]GOTO T,MATSUMOTO T,TAKAGI S.Distribution of the hepatic cysteamine dioxygenase activites in fish[J].Fisheries science,2001,67(6):1187-1189.
[12]TAPPAZ M,ALMARGHINI K,LEGAY F,et al.Taurine biosynthesis enzyme cysteine sulfinate decarboxylase(CSD)from brain:The long and tricky trail to identification[J].Neurochemical research,1992,17(9):849-859.
[13]GOTO T,MATSUMOTO T,MURAKAMI S,et al.Conversion of cysteate into taurine in liver of fish[J].Fisheries science,2003,69(1):216-218.
[14]UBUKA T,OKADA A,NAKAMURA H.Production of hypotaurine from L-cysteinesulfinate by rat liver mitochondria[J].Amino acids,2008,35(1):53-58.
[15]叶思平,陈悦娇,陈海光,等.高效液相色谱测定益智仁中的牛磺酸含量[J].食品科学,2013,34(10):204-206.
[16]陈申如,胡阳,倪辉,等.高效液相色谱法测定牡蛎中牛磺酸含量[J].中国食品学报,2013,13(2):193-198.
[17]柳小英,毛丽沙,鲍敏洁,等.高效液相色谱法测定海洋生物中牛磺酸的含量[J].安徽农业科学,2017,45(31):1-3,11.