Study On Detecting DAO In Serum With Speed-rate Method For Kit Preparation And The Clinical Application

Diamine oxidase (diamine oxidase;DAO, E.C.1.4.3.6) is the enzyme in the small intestine mucous membrane upper and the placental villi of human and mammal , which highly catalyzes oxidizing diamine (histamine, putrescine and cadaverin). DAO expresses highly in the fission cell. The necrosis cell falling off into the enteric cavity at the intestines mucous membrane causes the DAO activeness to reduce in intestines mucous membrane, and DAO activeness elevating in the intestines;DAO enters intercellular spaces of the intestines, and causes the DAO activeness to elevate in the blood plasma, so changing in the blood plasma DAO activeness may create in the situation to reflect the intestinal tract damage and the repair situation. Therefore, it will take the discussion of damage of intestinal tract function as an important target.In the placenta, higher diamine oxidase (DAO) was displayed. In the pregnant woman blood serum, the DAO activeness gets to advance. The determination of the DAO activeness in pregnancy woman blood serum has the vital significance to the gestation period monitor, in particular, for the threatened miscarriage history of the patient, determining the DAO activeness has become the new important target.This research for the first time using two kind of different reagents compositions carries on the coupling reaction in the different time. The blood serum DSO speed-rate kit was prepared successfully. The determination of DAO activeness may carries on in the human bloodserum in accuracy, sensibility and high speed. Its determination principle based on producing H2O2 and NH3 from tetramethylene-diamine by the DAO catalysis. NH3 and alpha ketoglutaric acid were catalysed by glutamic dehydrase (GLDH) to produce glutamic acid, which causes coenzyme I (NADH) to transform NAD+ in the reacting system, the OD drop value of NADH measured under the 340mm wave length may calculate the DAO activeness indirectly.The experiment showed that the DAO activeness was the biggest in buffer solution in 100 mmol L'1 and pH7.5, but the difference of the DAO activeness were not big between pH 7.0-9.0 . Comparing with the stability of other reagents, the buffer solution in 100 mmol L'1 and pH8.8 was selected for the best condition for determining DAO activeness.According to Michaelis-Menten equation, the Km value of DAO was determed to be 0.32 mmol L'1 Therefore, the best substrate concentration for measuring DAO activeness was determed as Km value 10 times of quantities( 3.2 mmol L-1). The DAO activeness scope inside connection was good in 0-100 U L"1. In batches CV respectively is 7.41, 4.10 and 3.11%;between batches CV respectively was 7.0, 4.60 and 3.26%. For the two kinds of blood plasma concentration, the average returns-ratios respectively were 97.9% and 98.4%. In blood plasma, the different DAO concentration were quite stable after being preserved for 7 days in -20 °C, 4 °C and 25 °C. The human blood plasma DAO activeness was not affected after going through 3 thaw-freeze cycles.The blood serum DAO activeness of 74 samples from healthy people as control experiment was 2.61±3.44 U L"1, and according to 95% credible sector, its diagnosis reference value is: DAO 9.49 U L'1, which is consistent to the short-term domestic literature reported [ 69, 124, 128 ]. For the 20 examples from the patients of I group, the blood serum DAO activeness respectively was 33.17±9.26 and 7.23±7.61 U L-1 before and after the treatment, the difference of comparison as healthy control was remarkable, P < 0.01. For the 20 examples from the patients of II group, the blood serum DAO activeness of the normally pre-natal and post-natal women were respectively 7.13H.41 and 3.32±2.47 U L"1. Comparison between the pre-natal and healthy control, the difference was remarkable, P < 0.05. For the post-natal and healthy control, the comparison difference was not remarkable, P > 0.05. In the threatened miscarriages patients of 12 examples of II group with diseases, the blood serum DAO activeness of the pre-natal and miscarriage were respectively 4.11 ±1.26 and 3.65±2.87 U L"1 . To compare the normal women in labor with those women before miscarriage, the difference was remarkable, P < 0.01;compared to the healthy control, the difference was not remarkable, P > 0.05. To compare the post-natal women with the normally healthy control, the difference was not remarkable, P > 0.05. For the sensitivities and the specificity of the group I with diseases and group II with, the positive forecast values and the negative forecast values respectively were 94.7%, 94.2%, 82.6%, 98.6% and 80.0%, 82.3%, 76.9%, 89.5%. In our experiment, the DSO speed-rate kit of blood serum wasdeveloped by using two kinds of different compositions of reagents, and carried on the coupling reaction in the different time. The method carried on the determination of DAO in human blood serum with good specialty , accuracy, and high speed.After the clinical preliminary examination, it was proved that the human blood serum DSO speed-rate kit may a ccurately carry on the determination to the DSO activeness of human blood serum. The determination of blood serum DAO activeness has the possibility to become the vital biochemical target for the intestinal tract damage, the intestinal tract barrier resulted from serious wound , the synthesis drafts of the MODS (multi- organs function disorder), and placenta monitor during gestation period, which has laid the foundation for the further clinical practice of blood serum DAO.