Gaudinski Julia B, Dawson Todd E, Quideau Sylvie, Schuur Edward A G, Roden John S, Trumbore Susan E, Sandquist Darren R, Oh Se-Woung, Wasylishen Roderick E
Department of Integrative Biology, University of California--Berkeley, 94720-3140, USA.
Anal Chem. 2005 Nov 15;77(22):7212-24. doi: 10.1021/ac050548u.
A number of operationally defined methods exist for pretreating plant tissues in order to measure C, N, and O isotopes. Because these isotope measurements are used to infer information about environmental conditions that existed at the time of tissue growth, it is important that these pretreatments remove compounds that may have exchanged isotopes or have been synthesized after the original formation of these tissues. In stable isotope studies, many pretreatment methods focus on isolating "cellulose" from the bulk tissue sample because cellulose does not exchange C and O isotopes after original synthesis. We investigated the efficacy of three commonly applied pretreatment methods, the Brendel method and two variants of the Brendel method, the Jayme-Wise method and successive acid/base/acid washes, for use on three tissue types (wood, leaves, roots). We then compared the effect of each method on C and O isotope composition (13C, 14C, 18O), C and N content, and chemical composition of the residue produced (using 13C nuclear magnetic resonance (NMR)). Our results raised concerns over use of the Brendel method as published, as it both added C and N to the sample and left a residue that contains remnant lipids and waxes. Furthermore, this method resulted in 18O values that are enriched relative to the other methods. Modifying the Brendel method by adding a NaOH step (wash) solved many of these problems. We also found that processed residues vary by tissue type. For wood and root tissues, the 13C NMR spectra and the 18O and 13C data showed only small differences between residues for the Jayme-Wise and modified Brendel methods. However, for leaf tissue, 13C NMR data showed that Jayme-Wise pretreatments produced residues that are more chemically similar to cellulose than the other methods. The acid/base/acid washing method generated 13C NMR spectra with incomplete removal of lignin for all tissues tested and both isotopic, and 13C NMR results confirmed that this method should not be used if purified cellulose is desired.
为了测量碳、氮和氧同位素，存在多种操作定义的方法用于预处理植物组织。由于这些同位素测量用于推断组织生长时存在的环境条件信息，因此这些预处理去除可能已交换同位素或在这些组织原始形成后合成的化合物非常重要。在稳定同位素研究中，许多预处理方法专注于从大量组织样本中分离“纤维素”，因为纤维素在原始合成后不会交换碳和氧同位素。我们研究了三种常用预处理方法（布伦德尔法以及布伦德尔法的两种变体、杰梅 - 怀斯法和连续酸/碱/酸洗）对三种组织类型（木材、叶子、根）的有效性。然后，我们比较了每种方法对碳和氧同位素组成（¹³C、¹⁴C、¹⁸O）、碳和氮含量以及产生的残留物的化学成分（使用¹³C核磁共振（NMR））的影响。我们的结果引发了对已发表的布伦德尔法使用的担忧，因为它既向样品中添加了碳和氮，又留下了含有残留脂质和蜡质的残留物。此外，该方法导致的¹⁸O值相对于其他方法有所富集。通过添加氢氧化钠步骤（洗涤）修改布伦德尔法解决了许多这些问题。我们还发现处理后的残留物因组织类型而异。对于木材和根组织，¹³C NMR光谱以及¹⁸O和¹³C数据显示，杰梅 - 怀斯法和改良布伦德尔法的残留物之间只有微小差异。然而，对于叶组织，¹³C NMR数据表明，杰梅 - 怀斯预处理产生的残留物在化学上比其他方法更类似于纤维素。对于所有测试组织，酸/碱/酸洗方法产生的¹³C NMR光谱显示木质素未完全去除，同位素和¹³C NMR结果均证实，如果需要纯化纤维素，则不应使用该方法。
