Laboratorio Bioquímica de Membrana, Programa de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Casilla 70005, Correo 7, Santiago, Chile.
Toxicon. 2010 Jan;55(1):135-44. doi: 10.1016/j.toxicon.2009.07.018. Epub 2009 Jul 24.
Paralytic shellfish toxins (PST) are a collection of over 26 structurally related imidazoline guanidinium derivatives produced by marine dinoflagellates and freshwater cyanobacteria. Glucuronidation of drugs by UDP-glucuronosyltransferase (UGT) is the major phase II conjugation reaction in mammalian liver. In this study, using human liver microsomes, the in vitro paralytic shellfish toxins oxidation and sequential glucuronidation are achieved. Neosaxitoxin (neoSTX), Gonyautoxin 3/2 epimers (GTX3/GTX2) and Saxitoxin (STX) are used as starting enzymatic substrates. The enzymatic reaction final product metabolites are identified by using HPLC-FLD and HPLC/ESI-IT/MS. Four metabolites from GTX3/GTX2 epimers precursors, three of neoSTX and two of STX are clearly identified after incubating with UDPGA/NADPH and fresh liver microsomes. The glucuronic-Paralytic Shellfish Toxins were completely hydrolysed by treatment with beta-glucuronidase. All toxin analogs were identified comparing their HPLC retention time with those of analytical standard reference samples and further confirmed by HPLC/ESI-IT/MS. Paralytic Shellfish Toxins (PST) were widely metabolized by human microsomes and less than 15% of the original PST, incubated as substrate, stayed behind at the end of the incubation. The apparent V(max) and Km formation values for the respective glucuronides of neoSTX, GTX3/GTX2 epimers and STX were determined. The V(max) formation values for Glucuronic-GTX3 and Glucuronic-GTX2 were lower than Glucuronic-neoSTX and Glucuronic-STX (6.8+/-1.9x10(-3); 8.3+/-2.8x10(-3) and 9.7+/-2.8x10(-3)pmol/min/mg protein respectively). Km of the glucuronidation reaction for GTX3/GTX2 epimers was less than that of glucuronidation of neoSTX and STX (20.2+/-0.12; 27.06+/-0.23 and 32.02+/-0.64microM respectively). In conclusion, these data show for the first time, direct evidence for the sequential oxidation and glucuronidation of PST in vitro, both being the initial detoxication reactions for the excretion of these toxins in humans. The PST oxidation and glucuronidation pathway showed here, is the hepatic conversion of its properly glucuronic-PST synthesized, and the sequential route of PST detoxication in human.
麻痹性贝类毒素（PST）是由海洋甲藻和淡水蓝藻产生的超过 26 种结构相关的咪唑啉胍基衍生物的集合。药物的葡萄糖醛酸化由 UDP-葡萄糖醛酰基转移酶（UGT）完成，是哺乳动物肝脏中主要的 II 相结合反应。在这项研究中，使用人肝微粒体，实现了麻痹性贝类毒素的体外氧化和顺序葡萄糖醛酸化。新石房蛤毒素（neoSTX）、GTX3/GTX2 差向异构体和扇贝毒素（STX）被用作起始酶促底物。通过 HPLC-FLD 和 HPLC/ESI-IT/MS 鉴定酶促反应的最终产物代谢物。用 UDPGA/NADPH 和新鲜肝微粒体孵育 GTX3/GTX2 差向异构体前体、neoSTX 的三种和 STX 的两种后，可明显鉴定出四种代谢物。用β-葡萄糖醛酸酶处理可完全水解葡萄糖醛酸-麻痹性贝类毒素。通过比较分析标准参考样品的 HPLC 保留时间和进一步通过 HPLC/ESI-IT/MS 确认，鉴定出所有毒素类似物。麻痹性贝类毒素（PST）被人微粒体广泛代谢，孵育为底物时，不到 15%的原始 PST 留在孵育结束时。确定了 neoSTX、GTX3/GTX2 差向异构体和 STX 各自葡萄糖醛酸化物的表观 V(max)和 Km 形成值。葡萄糖醛酸-GTX3 和葡萄糖醛酸-GTX2 的 V(max)形成值低于葡萄糖醛酸-neoSTX 和葡萄糖醛酸-STX（分别为 6.8+/-1.9x10(-3)、8.3+/-2.8x10(-3)和 9.7+/-2.8x10(-3)pmol/min/mg 蛋白）。GTX3/GTX2 差向异构体的葡萄糖醛酸化反应 Km 小于 neoSTX 和 STX 的葡萄糖醛酸化 Km（分别为 20.2+/-0.12、27.06+/-0.23 和 32.02+/-0.64μM）。总之，这些数据首次直接证明了 PST 的体外顺序氧化和葡萄糖醛酸化，这两种反应都是这些毒素在人体中排泄的初始解毒反应。这里显示的 PST 氧化和葡萄糖醛酸化途径是其适当的葡萄糖醛酸-PST 合成的肝转化，也是 PST 在人体中的解毒顺序途径。
