串联质谱技术对苯丙酮尿症杂合子的检测

Detection of phenylketonuria heterozygotes by tandem mass spectrometry

目的建立串联质谱技术测定血苯丙氨酸(Phe)和酪氨酸(Tyr)浓度方法,观察苯丙酮尿症(PKU)、PKU杂合子和正常对照组血Phe、Tyr浓度及其比值(Phe/Tyr)变化。方法研究对象为52例PKU患儿,152例PKU杂合子成人,160名正常成人作为对照。研究方法采用干血滤纸片法,血滤纸片经含已知浓度Phe和Tyr内标的甲醇萃取,盐酸正丁醇衍生后,用串联质谱仪分析。结果(1)样品回收率Phe在346μmol/L和485μmol/L浓度时分别为102%和100%,Tyr在276μmol/L和442μmol/L浓度时分别为103%和98%。(2)批内CVPhe为6.7%,Tyr为10.5%,批间CVPhe为12.2%,Tyr为9.9%。(3)PKU患者血Phe、Tyr浓度和Phe/Tyr值分别为(634.0±300.3)μmol/L、(41.9±16.3)μmol/L和16.5±9.9,PKU杂合子分别为(68.3±21.4)μmol/L、(40.1±11.8)μmol/L和1.7±0.4,对照组分别为(53.2±10.2)μmol/L、(43.6±9.5)μmol/L和1.2±0.2。PKU患者血Phe、Phe/Tyr值与PKU杂合子、对照组比较差异有统计学意义(P<0.01)。PKU杂合子与对照组比较,Phe、Phe/Tyr水平较高(均P<0.01),Tyr水平较低(P<0.01)。结论串联质谱技术能够准确测定干血滤纸片中Phe和Tyr浓度。PKU杂合子Phe浓度和Phe/Tyr值高于正常成人,但与正常人有明显的重叠。

Objective To establish a method to assay phenylalanine （Phe） and tyrosine（Tyr） by tandem mass spectrometry and to observe the changes of human blood Phe and Tyr levels and Phe/Tyr ratio in the subjects with phenylketonuria（PKU）, PKU heterozygotes and normal controls. Methods Fifty-two PKU patients, 152 PKU heterozygotes （parents of PKU patient） and 160 healthy adults （controls） were included in this study. Their blood filter paper were punched and extracted by methanol solution with stable isotope labeled internal standards, then derivatized with butanolic-HCI. After preparation, the samples were analyzed by tandem mass spectrometry. Results （ 1 ） The recovery of Phe were 102% and 100% on levels of 346μmol/L and 485μmol/L respectively. The recovery of Tyr were 103% and 98% on levels of 276μmol/L and 442μmol/L respectively. （2） The introday coefficient variation of Phe and Tyr were 6.7% and 10.5% , and the interday coefficient variation of Phe and Tyr were 12.2% and 9.9% respectively. （ 3 ） The levels of blood Phe, Tyr and Phe/Tyr were （ 634.0±300.3 ）μmol/ L, （41.9±16.3）μmol/L and 16.5±9.9 respectively in PKU patients, （ 68.3±21.4）μmol/L, （ 40.1±11.8 ）μmol/L and 1.7±0.4 respectively in PKU heterozygotes, and （53.2±10.2 ）μmol/L, （43.6±9.5）μmol/L and 1.2±0.2 respectively in health adults. The blood Phe level and Phe/Tyr were significantly higher in PKU patients than those in PKU heterozygotes and health adults （ all P〈0.01 ）. The blood Phe level and Phe/Tyr were higher in PKU heterozygotes than those in health adults （ both P〈0.01 ）. The blood Tyr level was lower in PKU heterozygotes than that in health adults （P〈0.01）. There were 4 cases of PKU heterozygotes whose level of Phe was higher than 120μmol/L （ the normal range 〈90μmol/L）. Conclusion Quantification of Phe and Tyr in blood filter spots by tandem mass spectrometry is fast and reliable. The levels of Phe and Phe/Tyr in PKU heterozygotes are higher than those in healthy adults, however there are partial overlapping.