Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

BACKGROUND: In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. RESULTS: To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. CONCLUSION: Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

研究背景：人体内存在四种碱性磷酸酶（alkaline phosphatase），每种酶均具有独特的组织分布特征。由于可通过简便方法检测其活性，目前已有大量研究数据报道了碱性磷酸酶活性变化与疾病之间的关联。例如，怀有21三体综合征（即唐氏综合征）胎儿的母亲，其外周血中性粒细胞中的碱性磷酸酶，在生化特性、免疫特性以及对部分特异性抑制剂的亲和力方面，均表现出显著差异。
研究结果：为分析上述差异，本研究将两种同工酶（非组织特异性碱性磷酸酶与胎盘型碱性磷酸酶）在杆状病毒感染的细胞中进行了表达。通过对这两种蛋白的对比分析，我们得以测定其变性动力学常数以及对两种抑制剂（L-对溴四咪唑与硫代磷酸酯）的敏感性，从而实现了两种酶的更精准区分。随后利用这些参数，我们对怀有21三体综合征胎儿与正常妊娠母亲的中性粒细胞内两种同工酶的比例进行了估算。研究发现，非妊娠女性中性粒细胞中的胎盘型同工酶占总活性的13%。正常妊娠状态下，该比例未出现显著升高。与之相反，怀有21三体综合征胎儿的妊娠母亲中，该比例可达总活性的60%至80%。
研究结论：相较于非组织特异性同工酶，怀有21三体综合征胎儿的母亲中性粒细胞内胎盘型同工酶的过表达，可解释为何此类细胞中碱性磷酸酶的特性与正常状态存在差异。将该研究成果应用于临床，可提升通过检测碱性磷酸酶活性筛查唐氏综合征的应用潜力。