Hipólito-Reis C, Dias P O, Martins M J
Serviço de Bioquimica, Faculdade de Medicina, Universidade do Porto, Portugal.
Scand J Clin Lab Invest. 1999 Dec;59(8):593-606. doi: 10.1080/00365519950185085.
The importance of separation and identification of serum alkaline phosphatase (ALP; E.C. 3.1.3.1) fractions/isoenzymes has been frequently reported. Each serum ALP fraction/isoenzyme quantitation has both practical and theoretical importance. In the present work, serum was collected from Wistar rats and, in identical experimental conditions, total serum ALP activity and serum ALP electrophoretic fractions/isoenzymes activities were quantified. Different results for both kinds of ALP activity were obtained when different buffers or mixture of these buffers (carbonate/bicarbonate; 2-amino-2-methyl-1-propanol/HCl; Veronal, sodium diethylbarbiturate/HCl), pH conditions (9.4 and 10.4) and substrates (alpha- and beta-naphthyl phosphates) were used. Higher total serum ALP activity was always observed with beta-naphthyl phosphate, independently of the buffer (or mixture of buffers) and pH used. Electrophoresis allowed the separation of two serum ALP fractions. Activity of both serum ALP electrophoretic fractions was always higher with beta-naphthyl phosphate, except with carbonate/bicarbonate pH 10.4. The effect of a change in pH was buffer- (or mixture of buffers) and substrate-dependent; the addition of a second buffer (to that previously used) was not always accompanied by an increase or decrease (of the same magnitude) in our results. The results obtained with different buffers (or mixture of buffers) were not identical with substrates and pH values. It is concluded that (i) from the same electrophoretic separation of serum ALP fractions/isoenzymes, different values for its activity can be obtained by changing the assay conditions used for ALP visualization (revelation, staining); (ii) the same assay conditions for quantitation of total serum ALP and serum ALP electrophoretic fractions/isoenzymes should be used; (iii) the choice of assay conditions should take into account the biochemical problem being studied in each case.
血清碱性磷酸酶（ALP；E.C. 3.1.3.1）各组分/同工酶的分离与鉴定的重要性已被频繁报道。每种血清碱性磷酸酶组分/同工酶的定量分析都具有实际和理论意义。在本研究中，从Wistar大鼠采集血清，并在相同的实验条件下，对血清碱性磷酸酶的总活性以及血清碱性磷酸酶电泳组分/同工酶的活性进行了定量分析。当使用不同的缓冲液或这些缓冲液的混合物（碳酸盐/碳酸氢盐；2-氨基-2-甲基-1-丙醇/盐酸；巴比妥缓冲液，二乙基巴比妥酸钠/盐酸）、pH条件（9.4和10.4）以及底物（α-和β-萘基磷酸酯）时，两种碱性磷酸酶活性得到了不同的结果。无论使用何种缓冲液（或缓冲液混合物）和pH值，使用β-萘基磷酸酯时总是观察到更高的血清碱性磷酸酶总活性。电泳可分离出两种血清碱性磷酸酶组分。除了在碳酸盐/碳酸氢盐pH 10.4的条件下，使用β-萘基磷酸酯时两种血清碱性磷酸酶电泳组分的活性总是更高。pH变化的影响取决于缓冲液（或缓冲液混合物）和底物；添加第二种缓冲液（相对于之前使用的缓冲液）并不总是会使我们的结果出现相同幅度的增加或减少。使用不同缓冲液（或缓冲液混合物）、底物和pH值所获得的结果并不相同。得出的结论是：（i）从血清碱性磷酸酶组分/同工酶的相同电泳分离中，通过改变用于碱性磷酸酶可视化（显色、染色）的检测条件，可以获得其活性的不同值；（ii）应使用相同的检测条件来定量血清碱性磷酸酶总活性以及血清碱性磷酸酶电泳组分/同工酶；（iii）检测条件的选择应考虑到每种情况下所研究的生化问题。
