关于脂肪酶分析

问：现在我用的南京建成的试剂盒来测脂肪酶，可是结果可以说是乱七八糟，一点也不稳定，这个问题也咨询过几位师兄，也有类似情况发生。不知道各位战友有没有碰到过这种情况？可有解决办法或者其他的测定方法？能否传授一下小弟？不胜感激！

答：我也用过南京建成的试剂盒，结果和你一样，看来试剂盒测脂肪酶还很不成熟。你可用常规方法（聚乙烯醇橄榄油乳化法），方法简单可靠，到处都能查到此方法。

答：乙烯醇的办法我试过，用国产试剂效果还是不好。建议用sigma的橄榄油，不过价格很贵。

实验方法如下：Lipase activity was assayed based on measurement of fatty acids release due to enzymatic hydrolysis of triglicerides in stabilized emulsion of olive oil (Borlongan, 1990). Assay was carried out by the addition of 1 ml of crude enzyme extract to 1 ml of stabilized lipase substrate in 1.5 ml of 0.1 M Tris–HCl buffer at pH 8.0. Mixture was incubated for 6 h at 37 jC, after which hydrolysis was stopped by addition of 3 ml of 95% ethyl alcohol. The mixture was then titrated with 0.01 N NaOH using 0.9% (w/v) thymolphthalein in ethanol as indicator. Blank determinations were conducted in a similar manner except that the crude enzyme extracts were introduced into the assay system after the 6-h incubation and immediately before titration. A unit of lipase activity (U) was defined as the volume of 0.01 N NaOH required to neutralize the fatty acidsreleased during the 6-h incubation with the substrate and after correction by the appropriate blank.

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