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目的建立血清磷酸二酯酶(PDE)活性测定的HPLC方法并探讨其在脑梗死患者的临床意义。方法分离293例脑梗死患者和148例对照组外周血白细胞,匀浆可溶部分为酶样品,以cAMP为反应底物,采用底物的减少来测定PDE的活性。色谱柱为Nova-Pak18C(3.9 mm×150 mm,4μm),流动相为甲醇-磷酸盐缓冲液(10:90,V/V);流速为0.8mL/min,柱温为室温;检测波长为254nm。结果PDE底物(cAMP、cGMP)和系列产物(5’AMP、5’GMP、腺苷、次黄嘌呤和腺嘌呤)分离良好,无干扰。cAMP和cGMP检测限分别为0.125μmoL/L和0.0625μmoL/L;平均回收率91.2%～102.8%;日内及日间RSD均<10%。脑梗死组外周血PDE活性为(13.805±3.121),对照组为(7.334±2.324),差异有统计学意义(P<0.001)。结论脑梗死患者PDE活性增高;HPLC法简单易行,能精确可靠地测定外周血白细胞的PDE活性。
Objective To establish a HPLC method for the determination of serum phosphodiesterase (PDE) activity and to explore its clinical significance in patients with cerebral infarction. Methods Peripheral blood leucocytes were isolated from 293 patients with cerebral infarction and 148 controls. The soluble fraction of homogenate was enzyme sample, and cAMP was used as the reaction substrate. The reduction of substrate was used to determine the activity of PDE. The column was Nova-Pak18C (3.9 mm × 150 mm, 4 μm) and the mobile phase was methanol-phosphate buffer (10:90, V / V). The flow rate was 0.8 mL / 254nm. Results PDE substrate (cAMP, cGMP) was separated from the products (5’AMP, 5’GMP, adenosine, hypoxanthine and adenine) without interference. The detection limits of cAMP and cGMP were 0.125μmoL / L and 0.0625μmoL / L respectively. The average recovery was 91.2% -102.8%. The intra-day and inter-day RSD were less than 10%. The PDE activity in peripheral blood was (13.805 ± 3.121) in cerebral infarction group and (7.334 ± 2.324) in control group, the difference was statistically significant (P <0.001). Conclusion The PDE activity in patients with cerebral infarction is increased. HPLC method is simple and easy to measure, and PDE activity of peripheral blood leucocytes can be determined accurately and reliably.