血尿琥珀酰丙酮检测在酪氨酸血症-Ⅰ型诊断中的应用

目的 探讨血、尿琥珀酰丙酮检测在酪氨酸血症-Ⅰ型诊断中的价值.方法 对临床疑似酪氨酸血症、血串联质谱检测酪氨酸水平增高的190例患儿,进行血串联质谱琥珀酰丙酮检测、尿气相色谱-质谱琥珀酰丙酮及有机酸检测.确诊条件为临床表现、血酪氨酸增高及血或尿琥珀酰丙酮增高.结果 确诊酪氨酸血症-Ⅰ型患儿11例,男9例,女2例,年龄2个月至6岁.患儿血琥珀酰丙酮显著升高( 14.2±7.8) μmol/L(参考值1～5μmol/L).其中7例患儿进行了尿气相色谱-质谱琥珀酰丙酮检测,4例阳性,3例阴性.血酪氨酸水平为(327.3±125.8) μmol/L(参考值为20 ～ 100μmol/L).临床表现均有肝肿大.结论 血或尿琥珀酰丙酮升高是诊断酪氨酸血症-Ⅰ型的最重要依据.串联质谱检测血琥珀酰丙酮较气相色谱-质谱检测尿琥珀酰丙酮对诊断酪氨酸血症-Ⅰ型更可靠,气相色谱-质谱尿琥珀酰丙酮检测可能导致假阴性.

Application of succinylacetone levels measurement in the blood and urine in the diagnosis of tyrosinemia type 1

Objective To establish the diagnostic method of tyrosinemia type 1 and evaluate its value,the succinylacetone levels in the blood of suspected patients with tyrosinemia were tested by tandem mass spectrometry, and the succinylacetone in the urine was tested by gas chromatography-mass spectrometry.Method A total of 190 patients suspected of having tyrosinemia,were tested by tandem mass spectrometry for measurement of the level of succinylacetone in the blood, and detected by gas chromatography-mass spectrometry for measurement of the level of succinylacetone and organic acid in the urine.The method of measuring the level of succinylacetone in blood by tandem mass spectrometry as follows: After the diameter of 3 mm dry blood spots were punched into wells of 96-well plate,100 μl 80％acetonitrile were added into each well,which contained hydrazine monohydrate and the internal standard of succinylacetone.The supernatant fluid were transferred to another 96-well plate and dried under heated nitrogen,after the plate was incubated for 30 min at 65 ℃.The residual hydrazine reagent was removed by addition of 100 μl methanol to each well and evaporated under heated nitrogen.The mobile phase ( 80％acetonitrile) was added to each well and 20 μ1 samples were tested by tandem mass spectrometry.The diagnostic terms were the clinical manifestation and the high level of succinylacetone in both blood and urine.Result Eleven patients were diagnosed as tyrosinemia type 1,with 9 males and 2 females.Their ages ranged from 2 months to 6 years.The succinylacetone levels in the blood of the patients were remarkably increased (7.26- 31.09 μmol/L),with an average of (14.2 ± 7.8 )μmol/L.Seven patients were tested for the level of succinylacetone in the urine by gas chromatography-mass spectrometry,and 4 were positive and 3 negative.Their tyrosine levels in the blood were 190 -543 μmol/L (Normal:20 - 100 μmol/L),with an average of (327.3 ± 125.8) μmol/L.All the patients presented the symptoms of hepatomegaly.Among them,9 patients died and 2 patients were improved after treatment.Conclusion The higher levels of succinylacetone in the blood or urine is a remarkable evidence for the diagnosis of tyrosinemia type 1.Determination of succinylacetone in the dry blood spots using tandem mass spectrometry was a good method for diagnosis of tyrosinemia type 1.To test succinylacetone in urine by gas chromatography-mass spectrometry may yield a false-negative result for tyrosinemia type 1.