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浙江大学学报(医学版)  2021, Vol. 50 Issue (4): 481-486    DOI: 10.3724/zdxbyxb-2021-0254
专题报道     
串联质谱法在过氧化物酶体病筛查中的应用
王燕敏1(),田国力1,*(),纪伟1,王四美2,张潇分1
1.上海市儿童医院 上海交通大学附属儿童医院新生儿筛查中心,上海 200040
2.上海市儿童医院 上海交通大学附属儿童医院神经内科,上海 200040
Very long chain acylcarnitines and lysophosphatidylcholines in screening of peroxisomal disease in children by tandem mass spectrometry
WANG Yanmin1(),TIAN Guoli1,*(),JI Wei1,WANG Simei2,ZHANG Xiaofen1
1. Neonatal Screening Center, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040, China;
2. Department of Neurology, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040, China
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摘要:

目的:探讨串联质谱法检测极长链酰基肉碱(VLCAC)和溶血磷脂酰胆碱(LPC)在过氧化物酶体病筛查中的价值。方法:选取2017年1月至2021年3月以发育迟缓等神经系统异常就诊于上海市儿童医院,根据临床症状、磁共振成像和基因检测结果明确诊断为X-连锁肾上腺脑白质营养不良(X-ALD)患儿14例和脑肝肾综合征(ZS)患儿4例。另选取同年龄段体检儿童200名为健康对照组。使用含稳定同位素内标的溶剂萃取所有对象干血斑标本中的VLCAC和LPC,直接采用串联质谱法检测二十碳酰基肉碱(C20)、二十二碳酰基肉碱(C22)、二十四碳酰基肉碱(C24)、二十六碳酰基肉碱(C26)、二十碳溶血磷脂酰胆碱(C20:0-LPC)、二十二碳溶血磷脂酰胆碱(C22:0-LPC)、二十四碳溶血磷脂酰胆碱(C24:0-LPC)和二十六碳溶血磷脂酰胆碱(C26:0-LPC)水平,并计算C24/C20、C24/C22、C26/C20、C26/C22、C24:0-LPC/C20:0-LPC、C24:0-LPC/C22:0-LPC、C26:0-LPC/C20:0-LPC、C26:0-LPC/C22:0-LPC比值。采用Kruskal-Wallis H检验和Mann-WhitneyU检验比较各组间VLCAC和LPC各指标检测值及比值,采用偏最小二乘法和变量投影重要度权重评分分析各指标对判断疾病的贡献度。结果:除C24:0-LPC/C20:0-LPC外,所有指标和比值在各组间差异均有统计学意义(P<0.05或P<0.01);X-ALD组与健康对照组、ZS组与健康对照组间,各指标有不同程度的差异,但X-ALD组与ZS组间差异无统计学意义(P>0.05);偏最小二乘法分析显示X-ALD和ZS组与健康对照组能够完全分离,C26的变量投影重要度值最大。结论:串联质谱法检测VLCAC和LPC可作为过氧化物酶体病筛查的方法,其中C26或可作为诊断敏感指标。

关键词: 过氧化物酶体病筛查串联质谱法极长链酰基肉碱溶血磷脂酰胆碱    
Abstract:

Objective:To investigate the value of very long chain acylcarnitine (VLCAC) and lysophosphatidylcholine (LPC) in screening of peroxisomal disease in children. Methods:Eighteen children with peroxisomal disease, including 14 cases of X-linked adrenoleukodystrophy (X-ALD group) and 4 cases of Zellweger syndrome (ZS group) diagnosed based on clinical symptoms, MRI and genetic tests were enrolled in the study; and 200 healthy children were selected as control group. Samples of dried blood spots were collected from all subjects, VLCAC and LPC in dried blood spots were extracted by solvent containing internal isotopic standards hexacosanoylcarnitine (2H3-C26) and C26:0 lysophosphatidylcholine (2H4-C26:0-LPC). The eicosanoylcarnitine (C20), docosanoylcarnitine (C22), tetracosanoylcarnitine (C24), hexacosanoylcarnitine (C26), C20:0 lysophosphatidylcholine (C20:0-LPC), C22:0 lysophosphatidylcholine (C22:0-LPC), C24:0 lysophosphatidylcholine (C24:0-LPC) and C26:0 lysophosphatidylcholine (C26:0-LPC) were detected by tandem mass spectrometry (MS/MS). The above 8 indicators and the ratios were compared among the groups using Kruskal-WallisH test and Mann-Whitney U test; the contribution of each index to the disease were analyzed by partial least square method. Results:Except C24:0-LPC/C20:0-LPC, there were significant differences in all indicators and ratios among all groups (P<0.05 orP<0.01). There were differences in most indicators and ratios between X-ALD group and the control group, as well as between ZS group and the control group, but there was no difference between the X-ALD group and the ZS group. PLS-DA analysis showed that the peroxisome disease group (including X-ALD group and ZS group) and the control group were able to be completely separated, and C26 had the highest variable importance for the projection (VIP) value.Conclusion:MS/MS detection of VLCAC and LPC can be used as a screening method for peroxisomal disease, and C26 may be a sensitive indicator for diagnosis.

Key words: Peroxisomal disease    Screening    Tandem mass spectrometry    Very long chain acylcarnitine    Lysophosphatidylcholine
收稿日期: 2021-04-26 出版日期: 2021-11-01
CLC:  R596  
基金资助: 上海市科学技术委员会科研计划(18441905100);上海市“重中之重”临床医学重点学科建设项目(2017ZZ02019);上海市卫生;计划生育委员会科研课题(2018ZHYL0233)
通讯作者: 田国力     E-mail: wangym@shchildren.com.cn;tiangl@shchildren.com.cn
作者简介: 王燕敏,主管技师,主要从事儿童遗传代谢病筛查工作;E-mail:wangym@shchildren.com.cn;https://orcid.org/0000-0003-1279-9422
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引用本文:

王燕敏,田国力,纪伟,王四美,张潇分. 串联质谱法在过氧化物酶体病筛查中的应用[J]. 浙江大学学报(医学版), 2021, 50(4): 481-486.

WANG Yanmin,TIAN Guoli,JI Wei,WANG Simei,ZHANG Xiaofen. Very long chain acylcarnitines and lysophosphatidylcholines in screening of peroxisomal disease in children by tandem mass spectrometry. J Zhejiang Univ (Med Sci), 2021, 50(4): 481-486.

链接本文:

http://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0254        http://www.zjujournals.com/med/CN/Y2021/V50/I4/481

组别

n

C20(μmol/L)

C22(μmol/L)

C24(μmol/L)

C26(μmol/L)

C20:0-LPC(μmol/L)

C22:0-LPC(μmol/L)

健康对照组

200

0.01(0.01,0.01)

0.01(0.01,0.01)

0.02(0.01,0.02)

0.01(0.01,0.01)

0.18(0.14,0.22)

0.15(0.12,0.17)

X-ALD组

14

0.02(0.015,0.02)*

0.01(0.01,0.02)*

0.10(0.08,0.11)*

0.11(0.09,0.12)*

0.39(0.27,0.60)*

0.27(0.25,0.37)*

ZS组

4

0.025(0.02,0.04)*

0.015(0.01,0.03)*

0.10(0.06,0.13)*

0.11(0.09,0.12)*

0.24(0.17,0.33)

0.16(0.12,0.21)

H

26.85

70.79

28.35

144.48

23.37

26.78

P

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

组别

n

C24:0-LPC(μmol/L)

C26:0-LPC(μmol/L)

C24/C20

C24/C22

C26/C20

C26/C22

健康对照组

200

0.38(0.34,0.42)

0.19(0.17,0.23)

1.50(1.00,2.00)

2.00(1.00,2.00)

2.00(1.00,2.00)

1.00(1.00,1.00)

X-ALD组

14

0.78(0.58,0.95)*

0.54(0.35,0.78)*

5.00(4.58,6.50)*

8.00(7.00,10.00)*

6.00(5.25,8.25)*

9.00(7.75,11.56)*

ZS组

4

0.62(0.55,0.68)*

0.68(0.61,0.89)*

2.75(1.83,5.63)

4.67(2.25,8.25)*

4.00(2.64,7.25)*

7.25(3.83,11.00)*

H

42.13

41.21

45.80

49.61

59.47

109.91

P

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

组别

n

C24:0-LPC/C20:0-LPC

C24:0-LPC/C22:0-LPC

C26:0-LPC/C20:0-LPC

C26:0-LPC/C22:0-LPC

健康对照组

200

1.00(0.82,1.25)

2.63(2.25,3.12)

0.52(0.39,0.67)

1.33(1.08,1.61)

X-ALD组

14

1.96(1.52,2.63)

2.32(2.2,3.22)

1.30(0.89,2.18)

1.77(1.00,2.50)

ZS组

4

2.42(2.12,2.95)

3.83(3.33,4.34)*

3.64(1.97,5.76)*

4.65(3.06,7.67)*

H

1.02

6.94

7.78

12.20

P

>0.05

<0.05

<0.05

<0.01

表 1  X-ALD组、ZS组与健康对照组极长链酰基肉碱和溶血磷脂酰胆碱检测结果比较
图 1  极长链酰基肉碱和溶血磷脂酰胆碱各指标筛查过氧化物酶体病的偏最小二乘法分析得分散点图X-ALD:X-连锁肾上腺脑白质营养不良;ZS:脑肝肾综合征.
图 2  极长链酰基肉碱和溶血磷脂酰胆碱各指标筛查过氧化物酶体病的变量投影重要度权重评分图C20:二十碳酰基肉碱;C22:二十二碳酰基肉碱;C24:二十四碳酰基肉碱;C26:二十六碳酰基肉碱;C20:0-LPC:二十碳溶血磷脂酰胆碱;C22:0-LPC:二十二碳溶血磷脂酰胆碱;C24:0-LPC:二十四碳溶血磷脂酰胆碱;C26:0-LPC:二十六碳溶血磷脂酰胆碱.
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