谷–丙转氨酶活性测定方法的改进

The Improvement of the Existing Experimental Scheme for the Determination of Serum ALT Activity

背景:目前,谷–丙转氨酶活性采用比色法测定。但在经典测定方案中,加入显色剂后,对照管中须加入与测量组等体积的谷丙转氨酶底物液,而对照管中先前加入的血清未灭活,可能具有酶活性,并与底物液发生额外的转氨基反应,造成阴性对照失真。目的:探究和了解经典谷–丙转氨酶活性测定实验方案的原理和流程,优化原有实验方案。方法:按经典方案进行操作,发现其由于对照管转氨酶不及时灭活可能对照失真的问题;改进方案验证,将测定管和对照管分别细分为灭活管与非灭活管,按经典方案进行操作,并对实验结果进行比较分析。结果:灭活与否显著影响对照管的光密度值,但不影响测定管光密度值。即灭活后的对照管光密度值明显小于未灭活的对照管,而灭活的测定管光密度值则几乎与未灭活的测定管相等。结论:经典实验在颜色反应前未对已有的转氨酶灭活,导致转氨基反应持续进行,对照管无法形成阴性对照。应该将原实验所有试管在颜色反应之前统一进行高温处理,以此将血清中的转氨酶及时灭活。

Background: At present, colorimetry was used to determine the activity of serum ALT. However, in this classical measurement scheme, we should add the same volume of ALT substrate solution to the control group as the experimental group after we add chromogenic agent. The serum ALT added before has not been inactivated, which means it may still have enzyme activity and catalyze addi-tional transamination with substrate solution at the same time, resulting in the distortion of the negative control. Objective: To explore and understand the principle and process of the classical experiment for the determination of ALT activity and optimize the original experimental scheme. Methods: We operated the classical scheme, and found that the inactivation of transaminase in the control tubes in time might cause the distortion of negative control. The improved scheme was set up to verify this conjecture, where the test tubes in the experimental group and the control group were both divided into inactivated tubes and non-inactivated tubes respectively. We operated the improved scheme in accordance with steps of the classical scheme and compared the results be-tween the two schemes. Results: The inactivation has a significant effect on the optical density of the control tubes, but not on that of the experiment tubes. That is, the optical density of the inactivated control tubes was significantly lower than that of the uninactivated control tubes in the original scheme, and the optical density of the inactivated experimental tubes was almost the same as that of the uninactivated experimental tubes in the original scheme. Conclusion: The classical experi-ment did not inactivate the existing ALT before the color reaction, which led to the continuous transamination so that the negative control could not be formed between the control group and ex-perimental group. All test tubes in the original experiment should be uniformly processed at high temperature before the color reaction so as to inactivate the serum ALT in time.