脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力
Summary
脂肪酸营养标记方法,即,将脂肪酸同化为整个分子, 并转移到没有或轻微修饰的消费者组织中, 这是由于小土壤无脊椎动物脂肪酸代谢的知识缺口所阻碍的。Isotopologue 分析作为一种宝贵的工具来解开营养的相互作用。
Abstract
脂肪酸 (FAs) 是食品网络生态学中有用的生物标志物, 因为它们通常被同化为一个完整的分子, 并转移到使用小的或没有修改的消费者组织中, 从而允许不同营养水平之间的膳食路由。然而, 由于对土壤动物脂质代谢的知识有限, 足总营养标记方法仍受到阻碍。本研究使用全标签棕榈酸 (13C16:0, 99 atom%) 作为示踪剂在脂肪酸代谢通路的两个广泛的土壤跳虫, Protaphorura fimata和Heteromurus 大足。为了研究这种前驱体的命运和代谢修饰, 提出了一种利用单离子监测的质谱法进行 isotopologue 分析的方法。此外, 还介绍了上游实验室饲养实验, 以及显性脂质组分 (中性脂质、磷脂) 的提取和甲基化, 以及相关的配方和计算。Isotopologue 分析不仅会产生从13c 标记为前体的脂肪酸中的总的13c 富集, 而且还产生超过母体离子质量的 isotopologues 的模式 (即, FA 分子离子m+) 每个标记为 FA 的一个或多个质量单位 (m+1, m+2, m+3,等等)。这一知识允许对完全消耗的 FA 的膳食路径与从头生物合成的比值进行结论。isotopologue 分析被认为是评价土壤动物脂肪酸代谢的有用工具, 以解开营养的相互作用。
Introduction
在一个神秘的栖息地如土壤, 营养关系是难以解决, 并进一步限制小规模的动物。在过去的十年中, 生物化学生态学取得了进展, 特别是在使用脂肪酸作为生物标志物, 以确定田间条件下土壤动物的饲养策略1,2,3。这是基于一个事实, 即从资源中脂肪酸可以被纳入到消费组织作为整个分子, 一个过程称为膳食路由4。从真菌到线虫到跳虫 5, 已经报告了三多种营养水平的脂肪酸转移。最近, 捕食动物区系被认为是6,7和第一次关于脂肪酸的评论作为土壤食物网的营养标记被发布了8,9。
通过脂肪酸稳定同位素探针 (FA-SIP) 获得了更详细的营养相互作用信息。在饮食和消费者中脂肪酸的13c/12c 比值的确定可以归因于二进制链接和估计伴生的碳流动, 并且被使用了在地球、淡水和海洋食物网10,11 ,12,13。基本的假设是, 膳食路由脂肪酸不受酶过程的影响;因此, 它们的13C 信号,即 13c/12c 比脂肪酸, 在消费者中类似于饮食1。然而, 在水生系统中报告了食物链的13C 签名逐渐枯竭, 从而阻碍了在营养研究中广泛应用 FA SIP14,15,16。此外, 陆地食物网中大多数无脊椎动物的脂代谢知识仍然有限。
了解消费者的脂质代谢通路, 对于利用营养标记脂肪酸作为测定食品网络生态学中定量碳流量的手段是至关重要的。考虑到这一点, 13C-isotopologue 分析, 原则上可以应用于对任何生物系统的碳代谢的研究17, 这是一个有希望的方法。在引入13c 标记的碳基板之后, 在新陈代谢网络中的13C 的分布是可追踪的, 因为在消费者中产生的代谢产物显示了特定的 isotopologue 分布。这可以通过定量的核代谢共振光谱学18,19或质谱20,21来评估, 后者在生物样品中偏爱低生物量由于其较高的灵敏度。
虽然 isotopologue 分析已成功应用于氨基酸, 并提供了对细菌病原体的体内碳代谢的洞察力17,22,23, 它的实施在脂肪酸已经落后了。第一次详细分析了稳定同位素标签前体脂肪酸的命运, 其膳食路由或降解通过β氧化, 在土壤无脊椎动物的消费者, 最近进行了门泽尔et 等。24. 在这里, 给出了与13C 标记脂肪酸结合实验的方法学基础, 并对常见的土壤无脊椎动物 (跳虫) 中的关键子代进行了 isotopologue 分析。这些 microarthropods 是一个很好的模型组, 因为它们构成了土壤食物网的重要组成部分, 并对其营养标记脂肪酸8,25进行了良好的调查。
了解消费者的脂质代谢通路, 对于利用营养标记脂肪酸作为测定食品网络生态学中定量碳流量的手段是至关重要的。本协议给出了实验室饲养实验的设计和建立, 以及从跳虫中提取和甲基化显性脂质分数 (中性脂质、磷脂) 的生物化学程序。通过质谱分析, 说明了脂肪酸的 isotopologue 组成, 并描述了相关的公式和计算。此过程的结果是: (i) isotopologues 超过父离子质量的比率 (即,脂肪酸分子离子 M+) 由一个或多个质量单位 (m+1, m+2, M+3,等) 和 (二) 总体13c. 从13C 标记的前驱体中提炼出的脂肪酸。虽然使用跳虫, 这种方法一般可以适用于任何其他捕食者-猎物互动的前提下, 这些是可培养在足够数量的控制条件下, 以确保成功的标签摄取和随后验证。
Protocol
Representative Results
Discussion
Isotopologue 分析
对 FAs 中13C 分布的数量方面的详细分析需要尖端技术来分配食物网中的碳分割。目前的工作使用 isotopologue 分析来评估热带相互作用的共同 FA 生物标志物的13c/12c 比值。采用液相色谱法 (LC) 对氨基酸进行分析, 并应用于致病细菌的碳代谢研究17,23。直到最近, isotopologue 的分析进一步…
Acknowledgements
门泽尔和 l. Ruess 的财政支持由德意志 Forschungsgemeinschaft (汝 RU780/11-1) 感激地承认。r. Nehring 由汝 780/10-1 资助。最后, 我们非常感谢榛 Ruvimbo Maboreke 博士校对我们的手稿。
Materials
| neoLab-Round jars | neoLab | 2-1506 | 69 x 40 mm, 10 pacs/pack |
| Charcoal activated | Carl Roth | X865.1 | p.a., powder, CAS No. 7440-44-0 |
| Alabaster Dental | RÖHRICH-GIPSE | — | http://www.roehrich-gipse.de/dentalgipse.php |
| Chloroform | Carl Roth | 7331.1 | HPLC ≥ 99,9 % |
| Methanol | Carl Roth | P717.1 | HPLC ≥ 99,9 % |
| Hexan | Carl Roth | 7339.1 | HPLC ≥ 98 % |
| tert-Butyl methyl ether (MTBE) | Carl Roth | T175.1 | HPLC ≥ 99,5 % |
| Aceton | Carl Roth | 7328.2 | HPLC ≥ 99,9 % |
| NaOH | Carl Roth | 6771.1 | p.a. ≥99 %, in pellets |
| di-Natriumhydrogenphosphat | Carl Roth | P030.1 | p.a. ≥99 % , water free |
| Na-dihydrogenphosphat Dihydrat | Carl Roth | T879.1 | p.a. ≥99 % |
| Hypochloric acid (6 N) | VWR International | 26,115,000 | AVS TITRINORM vol. solution |
| Bond Elut (Columns) | Agilent Tech. | 14102037 | HF Bond Elut-SI, 500 mg, 3 mL, 50/PK |
| Präparatengläser Duran | Glasgerätebau Ochs | 135215 | Ø 16 x 100 mm, plus screw cap with handy knurl and integrated PTFE/silicone gasket |
| Supelco 37 Component FAME Mix | Sigma-Aldrich | 47885-U Supelco | 10 mg/mL in methylene chloride, analytical standard |
| FlowMesh | Carl Roth | 2796.1 | Polypropylene mesh, approximately 0.3 mm thick, with 1 mm strand spacing |
| Bacterial Acid Methyl Ester (BAME) Mix | Sigma-Aldrich | 47080-U Supelco | 10 mg/mL in methyl caproate, analytical standard |
| Methyl nonadecanoate | Sigma-Aldrich | 74208 | analytical standard ≥ 98.0 % |
| Hexadecanoic acid-1-13C (Palmitic) | Larodan Fine Chemicals | 78-1600 | GC ≥ 98.0 % (13C: 99.0 %) |
| RVC 2-25 CDplus | Martin Christ Gefrier-trocknungsanlagen | Compact benchtop midi concentrator | |
| Alpha 2-4 LDplus | Martin Christ Gefrier-trocknungsanlagen | Drying manifold | |
| MZ 2C NT | Vacuubrand GMBH | Vacuum pump | |
| Roto-Shake Genie | Scientific Industries | Combined rocking and rotating device | |
| XP64 Micro Comparator | Mettler Toledo | Super high precision balance | |
| GC-System 7890A | Agilent Tech. | Gas chromatograph | |
| 7000 GC/MS Triple Quad | Agilent Tech. | Triple Quad mass spectrometer | |
| 7683B Series Injector | Agilent Tech. | Sample injector | |
| Heraeus Multifuge 3SR+ | Thermo Scientific | Centrifuge with 10 ml tube rotor |
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