Institute of Food Chemistry, University of Muenster, Muenster, Germany.
J Agric Food Chem. 2011 Jun 8;59(11):6018-24. doi: 10.1021/jf200943n. Epub 2011 May 16.
The sphingolipid composition of food as well as of physiological samples has received considerable interest due to their positive biological activities. This study quantified the total amount of sphingomyelin (SM) in 20 human breast milk samples from healthy volunteers and determined the structures of SM by detailed mass spectrometric studies in combination with enzymatic cleavage. The quantification of SM was performed by hydrophilic interaction liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (HILIC-HPLC-ESI-MS/MS) measuring the characteristic fragment ion of the phosphorylcholine group at m/z 184.2 and by using hexanoylsphingomyelin (C6-SM) and heptadecanoylsphingomyelin (C17-SM) as internal standards. The structures of SM species were identified after enzymatic cleavage with alkaline sphingomyelinase (SMase) to the corresponding ceramides. Structure elucidation of the sphingoid base and fatty acid backbone was performed by reversed-phase HPLC-ESI-MS/MS. The method includes the sphingoid bases dihydrosphingosine (d18:0), sphingosine (d18:1(Δ4)), 4,8-sphingadienine (d18:2(Δ4,8)), 4-hydroxysphinganine (phytosphingosine (t18:0)), and 4-hydroxy-8-sphingenine (t18:1(Δ8)) and fatty acids with even-numbered carbon atoms (C12-C26) as well as their (poly)unsaturated and monohydroxylated analogues. The total amount of SM in human breast milk varied from 3.87 to 9.07 mg/100 g fresh weight. Sphingosine (d18:1) was the predominant sphingoid base, with 83.6 ± 3.5% in human breast milk, followed by 4,8-sphingadienine (d18:2) (7.2 ± 1.9%) and 4-hydroxysphinganine (t18:0) (5.7 ± 0.7%). The main SM species contained sphingosine and palmitic acid (14.9 ± 2.2%), stearic acid (12.7 ± 1.5%), docosanoic acid (16.2 ± 3.6%), and tetracosenoic acid (15.0 ± 3.1%). Interestingly, the fatty acid composition of SM species in this study differs from the total fatty acids in human breast milk, and the fatty acids are not consistently distributed among the different sphingoid bases.
由于其积极的生物学活性，食物以及生理样本中的神经鞘脂组成受到了相当大的关注。本研究定量测定了 20 份健康志愿者母乳样本中的总鞘磷脂 (SM) 含量，并通过详细的质谱研究结合酶切确定了 SM 的结构。SM 的定量通过亲水相互作用液相色谱-电喷雾串联质谱法 (HILIC-HPLC-ESI-MS/MS) 进行，该方法测量磷酰胆碱基团的特征碎片离子 m/z 184.2，并使用己酰基神经鞘氨醇 (C6-SM) 和十七酰基神经鞘氨醇 (C17-SM) 作为内标。SM 物种的结构在碱性神经鞘磷脂酶 (SMase) 作用下被切割成相应的神经酰胺后进行鉴定。通过反相 HPLC-ESI-MS/MS 对神经鞘氨醇碱基和脂肪酸主链进行结构阐明。该方法包括神经鞘氨醇碱基二氢神经鞘氨醇 (d18:0)、神经醇 (d18:1(Δ4))、4,8-二氢神经鞘氨醇 (d18:2(Δ4,8))、4-羟基神经鞘氨醇 (植物神经鞘氨醇 (t18:0)) 和 4-羟基-8-神经鞘氨醇 (t18:1(Δ8)) 以及偶数碳原子 (C12-C26) 的脂肪酸及其 (多) 不饱和和单羟基化类似物。人乳中 SM 的总量从 3.87 到 9.07 mg/100 g 鲜重不等。神经醇 (d18:1) 是主要的神经鞘氨醇碱基，占人乳的 83.6±3.5%，其次是 4,8-二氢神经鞘氨醇 (d18:2) (7.2±1.9%) 和 4-羟基神经鞘氨醇 (t18:0) (5.7±0.7%)。主要的 SM 物种含有神经醇和棕榈酸 (14.9±2.2%)、硬脂酸 (12.7±1.5%)、二十二烷酸 (16.2±3.6%) 和二十四烷酸 (15.0±3.1%)。有趣的是，本研究中 SM 物种的脂肪酸组成与母乳中的总脂肪酸不同，脂肪酸在不同的神经鞘氨醇碱基之间的分布并不一致。
