| 常韶燕,李佰一,姚秀英,刘鑫丽,王 理.组织叶酸检测方法的建立及应用[J].,2019,19(7):1246-1250 |
| 组织叶酸检测方法的建立及应用 |
| Establishment and Application of Tissue Folic Acid Detection Method |
| 投稿时间：2018-11-28 修订日期：2018-12-23 |
| DOI：10.13241/j.cnki.pmb.2019.07.009 |
| 中文关键词: 叶酸 Tris-base盐溶液 PBS Lysis |
| 英文关键词: Folic acid Tris-base salt solution PBS Lysis |
| 基金项目:北京市属医学科研院所公益发展改革与发展专项（京医研2016-4）；北京市医院管理局儿科学科协同发展中心专项（XTZD20180402） |
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| 中文摘要: |
| 摘要 目的：采用目前常用的化学发光法检测组织叶酸，探索不同叶酸前处理，寻求合适的检测组织叶酸的实验方法。并将优化好的方法应用于叶酸缺乏小鼠模型的检测。方法：通过化学发光法检测组织叶酸含量，首先将一定量的组织放入合适的buffer中，匀浆器匀浆后，通过超声或煮沸处理样本，离心后，得到上清样本，建立标曲和质控后，进行上机测定。该方法中我们通过探讨不同的buffer（包括PBS、Lysis和Tris-base盐溶液）处理组织叶酸，和不同的处理条件（包括是否煮沸及煮沸时间、样本处理时间等），探讨该方法检测组织叶酸最优化条件；并应用该方法进行小鼠叶酸缺乏模型中各组织叶酸含量的检测。结果：我们选择雄鼠肝组织进行叶酸检测，分别用PBS、Lysis和Tris-base盐溶液进行样本前处理，结果发现同样的处理条件下，如不煮沸条件下，Tris-base盐溶液（38.72 ng/mg）>PBS（15.68 ng/mg）>Lysis（11.9 ng/mg），提示Tris-base盐溶液可能有助于叶酸的稳定，防止降解。同时，我们探讨煮沸对叶酸检测结果的影响，结果发现同一种前处理下（如Tris-base），不煮沸（38.72 ng/mg）>煮沸1分钟（36.36 ng/mg）>煮沸3分钟（33.28 ng/mg）>煮沸5分钟（30.72 ng/mg），说明叶酸含量随着煮沸时间的延长逐渐降低。此外，我们还对叶酸检测结果的重复性和稳定性进行了评估，结果发现不煮沸条件下，叶酸结果重复性很好，但随着时间延长稳定性会逐渐下降。因此，我们选择了Tris-base盐溶液进行前处理，条件选择为不煮沸和立即检测来减少因样本处理问题产生的误差。我们用该方法检测了叶酸缺乏小鼠模型的各组织叶酸含量，结果发现肝组织18.81 ng/mg降为8.46 ng/mg，脑组织从0.37 ng/mg下降为0.19 ng/mg，脾脏组织从1.53 ng/mg下降为0.26 ng/mg，肺组织从0.47 ng/mg下降为0.13 ng/mg，肾脏组织从2 ng/mg下降为0.9 ng/mg，心脏组织从0.33 ng/mg下降为0.06 ng/mg说明饮食叶酸是体内叶酸的主要来源, 其缺乏可能导致多种组织叶酸代谢异常。结论：组织叶酸化学发光法检测条件优化为Tris-base盐溶液进行前处理，条件选择为不煮沸，并将样本立即进行检测。我们用该方法检测小鼠叶酸缺乏模型发现低叶酸饮食能显著降低各组织的叶酸含量，可能对生长发育造成潜在的影响。 |
| 英文摘要: |
| ABSTRACT Objective: To find an appropriate experimental method for detecting folic acid in tissues, we selected the method of chemiluminescence (CL) and explored different pretreatments of folic acid. Furthermore, we also explored the application of this method in folic acid deficiency mice model. Methods: The folic acid content in tissues was detected by chemiluminescence method. Firstly, a cer- tain amount of tissues were put into a suitable buffer. After homogenizing, the samples were treated by ultrasound or boiling. The super- natant samples were obtained after centrifugation.The samples were determined by the AccessII analyisis platform after the establishment of curvature and quality control. In this method, we investigated the optimal conditions for detecting folic acid in tissues by different buffer treatments (including PBS, Lysis and Tris-base salt solution) and different treatment conditions (including boiling time, sample processing time, etc.). Using this method, we detection of folic acid content of various tissues in the folic acid-deficienct mouse model. Results: Liver tissues of male mice were selected and pretreated with PBS, Lysis and Tris-base salt solution respectively. The results showed that under the same treatment conditions, without boiling, the folic acid content in Tris-base salt solution group (38.72 ng/mg) was the highest than in PBS (15.68 ng/mg) and Lysis (11.9 ng/mg) suggesting that Tris-base salt solution may contribute to the stability of folic acid and prevent its degradation. Meanwhile, we investigated the effect of boiling on folic acid concentration. The results showed that under the same pretreatment (such as Tris-base), the content of folic acid gradually decreased with the prolongation of boiling time，with no boiling of 38.72 ng/mg, boiling for 1 minute of 36.36 ng/mg, boiling for 3 minutes of 33.28 ng/mg, boiling for 5 minutes of 30.72 ng/mg. In addition, we also evaluated the repeatability and stability of folic acid test results. The results showed that the repeatability of folic acid test was good under the condition of no boiling, but the stability would gradually decrease with time. Therefore, we used Tris-base salt solution for pretreatment, under the condition of non-boiling and immediate detection to reduce the error caused by sample preprocessing. Folic acid content in tissues of folic acid deficient mice was determined by this method. The results showed that folic acid content in tissues of folic acid deficient mice was significantly lower than that of normal mice, with 18.81 ng/mg decreasing to 8.46 ng/mg of liver, 0.37 ng/mg decreasing to 0.19 ng/mg of brain, 1.53 ng/mg decreasing to 0.26 ng/mg of spleen, 0.47 ng/mg decreasing to 0.13 ng/mg of lung, 2 ng/mg decreasing to 0.9 ng/mg of kindey, 0.33 ng/mg decreasing to 0.06 ng/mg of heart, indicating that folic acid in diet was the main source of folic acid in vivo and folic acid deficiency may cause folic acid metabolism abnormalities in a variety of tissue. Conclusion: The chemiluminescence method detected tissue folic acid is optimized to pretreat with Tris-base salt solution, and the condition is not boiling, and the samples are detected immediately. Using the method, we detected the tissue of folic acid-deficient mouse model and found that low folic acid diet can significantly reduce the folic acid content in various tissues, which may have a poten- tial impact on growth and development. |
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