Larsen Erik H, Lobinski Ryszard, Burger-Meÿer Karin, Hansen Marianne, Ruzik Rafal, Mazurowska Lena, Rasmussen Peter Have, Sloth Jens J, Scholten Olga, Kik Chris
Department of Food Chemistry, Danish Institute for Food and Veterinary Research, 2860 Søborg, Denmark.
Anal Bioanal Chem. 2006 Jul;385(6):1098-108. doi: 10.1007/s00216-006-0535-x. Epub 2006 Jun 13.
The scope of the work was to investigate the influence of selenate fertilisation and the addition of symbiotic fungi (mycorrhiza) to soil on selenium and selenium species concentrations in garlic. The selenium species were extracted from garlic cultivated in experimental plots by proteolytic enzymes, which ensured liberation of selenium species contained in peptides or proteins. Separate extractions using an aqueous solution of enzyme-deactivating hydroxylamine hydrochloride counteracted the possible degradation of labile selenium species by enzymes (such as alliinase) that occur naturally in garlic. The selenium content in garlic, which was analysed by ICP-MS, showed that addition of mycorrhiza to the natural soil increased the selenium uptake by garlic tenfold to 15 microg g(-1) (dry mass). Fertilisation with selenate and addition of mycorrhiza strongly increased the selenium content in garlic to around one part per thousand. The parallel analysis of the sample extracts by cation exchange and reversed-phase HPLC with ICP-MS detection showed that gamma-glutamyl-Se-methyl-selenocysteine amounted to 2/3, whereas methylselenocysteine, selenomethionine and selenate each amounted to a few percent of the total chromatographed selenium in all garlic samples. Se-allyl-selenocysteine and Se-propyl-selenocysteine, which are selenium analogues of biologically active sulfur-containing amino acids known to occur in garlic, were searched for but not detected in any of the extracts. The amendment of soil by mycorrhiza and/or by selenate increased the content of selenium but not the distribution of detected selenium species in garlic. Finally, the use of two-dimensional HPLC (size exclusion followed by reversed-phase) allowed the structural characterisation of gamma-glutamyl-Se-methyl-selenocysteine and gamma-glutamyl-Se-methyl-selenomethionine in isolated chromatographic fractions by quadrupole time-of-flight mass spectrometry.
该研究工作的范围是调查硒酸盐施肥以及向土壤中添加共生真菌（菌根）对大蒜中硒及硒形态浓度的影响。通过蛋白水解酶从试验田中种植的大蒜中提取硒形态，该酶可确保释放肽或蛋白质中所含的硒形态。使用失活酶的盐酸羟胺水溶液进行单独提取，可抵消大蒜中天然存在的酶（如蒜氨酸酶）对不稳定硒形态可能造成的降解。通过电感耦合等离子体质谱法（ICP-MS）分析大蒜中的硒含量，结果表明，向天然土壤中添加菌根可使大蒜对硒的吸收增加至10倍，达到15微克/克（干重）。用硒酸盐施肥并添加菌根可使大蒜中的硒含量大幅增加至约千分之一。通过阳离子交换和反相高效液相色谱法（HPLC）结合ICP-MS检测对样品提取物进行平行分析，结果表明，γ-谷氨酰基-Se-甲基-硒代半胱氨酸占2/3，而甲基硒代半胱氨酸、硒代蛋氨酸和硒酸盐在所有大蒜样品中各占色谱分析总硒的百分之几。寻找了大蒜中已知的具有生物活性的含硫氨基酸的硒类似物Se-烯丙基-硒代半胱氨酸和Se-丙基-硒代半胱氨酸，但在任何提取物中均未检测到。通过菌根和/或硒酸盐改良土壤增加了大蒜中硒的含量，但未改变所检测到的硒形态的分布。最后，使用二维HPLC（尺寸排阻后接反相），通过四极杆飞行时间质谱法对分离的色谱馏分中的γ-谷氨酰基-Se-甲基-硒代半胱氨酸和γ-谷氨酰基-Se-甲基-硒代蛋氨酸进行了结构表征。
