液相色谱串联质谱检测尿黏多糖在黏多糖贮积症患者诊断与随访中的应用

doi:10.12372/jcp.2024.24e0050

摘要/Abstract

摘要：

目的运用液相色谱串联质谱（LC-MS/MS）技术建立尿液中硫酸软骨素(CS)、硫酸皮肤素(DS)和硫酸乙酰肝素(HS)含量的检测方法，探究该方法在黏多糖贮积症（MPS）患者诊断及治疗随访中的应用。方法收集20例MPS患儿及37例正常儿童尿液样本，测定尿液中CS、DS和HS等浓度。尿样氮气吹干，盐酸甲醇衍生化，再次氮气吹干，复溶后LC-MS/MS分析；评价方法的线性、定量限、精密度、加样回收率和基质效应。结果 LC-MS/MS检测CS、DS和HS的线性均大于0.99。CS、DS和HS的定量下限依次为：0.5、1.0和1.0 mg/L。批内不精密度为1.9％~10.4％，批间不精密度为2.6％~9.8％。加标回收率为85.6％~110.4％，相对基质效应为84.9%~115.5%。CS、DS和HS在正常儿童尿液中呈正态分布，以 x+1.64 SD计算正常儿童尿液中的参考区间上限，分别为16.5、1.8、1.4 mg/mmol。通过分析MPS患儿尿液黏多糖特点，LC-MS/MS法能有效检出MPS I、MPS Ⅱ、MPS Ⅲ和MPS Ⅵ等MPS患儿。已接受造血干细胞移植的MPS Ⅰ和MPS Ⅱ患儿尿DS、HS水平降低。结论 LC-MS/MS检测尿黏多糖性能良好，有望用于MPS患者的精准诊断和治疗随访监测。

关键词: 液相色谱串联质谱, 黏多糖, 黏多糖贮积症

Abstract:

Objective To establish a method to determine chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS), and to investigate the application of urine glycosaminoglycans detection in the diagnosis and follow-up of mucopolysaccharidosis (MPS) patients. Methods Twenty urine samples from MPS patients and 37 urine samples from normal children were collected, and glycosaminoglycans levels in these samples were determined. Urine samples were added and dried under nitrogen. Then the samples were derivatized with methanol hydrochloride and dried under nitrogen. After reconstituted, the glycosaminoglycans were analyzed by LC-MS/MS. The linearity, limit of quantification, precision, spiked recoveries, and matrix effect were evaluated. Results The linearity of CS, DS and HS by LC-MS/MS was>0.99. The limits of quantification for CS, DS and HS were 0.5 mg/L, 1 mg/L and 1 mg/L, respectively; the intra-day imprecision ranged from 1.9% to 10.4%, and the inter-day imprecision ranged from 2.6% to 9.8%; and the recovery ranged from 85.6% to 110.4%, the relative matrix effect ranged from 84.9% to 115.5%. CS, DS and HS in urine samples of normal children were normally distributed, then the upper limit of reference interval were preliminarily determined by mean+1.64 SD, which were 16.5 mg/mmol, 1.8 mg/mmol and 1.4 mg/mmol, respectively. Urine GAGs of MPS patients were analyzed, the LC-MS/MS method was effective in detecting MPSⅠ, MPSⅡ, MPSⅢ and MPSⅥ patients. Urine samples of MPSⅠ or MPSⅡ patients who had hematopoietic stem cell transplantation showed reduced DS and HS concentration. Conclusion LC-MS/MS has good performance in detecting urinary mucopolysaccharides and is expected to be used for accurate diagnosis and treatment follow-up monitoring of MPS patients.

Key words: liquid chromatography-tandem mass spectrometry, glycosaminoglycans, mucopolysaccharidosis

占霞, 高晓岚, 季文君, 常思宇, 刘丹, 张惠文. 液相色谱串联质谱检测尿黏多糖在黏多糖贮积症患者诊断与随访中的应用[J]. 临床儿科杂志, 2024, 42(5): 399-406.

ZHAN Xia, GAO Xiaolan, JI Wenjun, CHANG Siyu, LIU Dan, Zhang Huiwen. Detection of urine glycosaminoglycans by liquid chromatography-tandem mass spectrometry in the diagnosis and follow-up of patients with mucopolysaccharidosis[J]. Journal of Clinical Pediatrics, 2024, 42(5): 399-406.

图/表 5

表1

MPS患者的尿液黏多糖质谱检测和电泳结果分析"

样本	性别	年龄 /岁	疾病诊断	临床表现	基因	变异位点	CS/肌酐/ mg·mmol^-1	DS/肌酐/ mg·mmol^-1	HS/肌酐/ mg·mmol^-1	电泳条带
正常儿童组 (n=37)	-	-	-	-	-	-	7.7±5.4	0.8±0.6	0.6±0.4	?
P1	男	1.3	MPS I	粗糙面容、角膜混浊、脊柱后突	IDUA	c.1402+2T>G/ c.1045G>C, p.D349H	42.1	12.9	51.0	CS、DS
P2	女	4.0	MPS I	粗糙面容、关节僵硬、肝大	-	-	11.6	26.9	58.4	CS、DS
P3	男	2.0	MPS I	粗糙面容、角膜混浊、脊柱后突、关节僵硬	-	-	71.6	142.4	125.9	CS、DS
P4	男	6.1	MPS I +HSCT	粗糙面容、爪形手、关节僵硬改善	IDUA	c.1037T>G, p.L346R/ c.1210G>T, p.E404*	10.1	2.4	1.9	无条带
P5	男	6.0	MPS I +HSCT	智力落后、四肢畸形、脊柱后突	IDUA	c.536C>G, p.Y179R/ c.536C>G, p.Y179R	10.2	4.1	1.9	无条带
P6	男	4.9	MPS I +HSCT	脊柱侧弯、粗糙面容、角膜轻微浑浊	-	?	6.5	2.4	1.5	无条带
P7	男	5.0	MPS Ⅱ	多动、关节僵硬、毛发重、心脏累及	IDS	c.268T>G, p.S90P	10.8	23.7	140.0	DS、CS、 HS
P8	男	5.0	MPS Ⅱ	粗糙面容、关节僵硬、骨骼畸形、智力落后、心脏累及	-	?-	10.7	24.3	44.2	DS、CS
P9	男	5.8	MPS Ⅱ	智力落后、关节僵硬、肝大	IDS	c.1006+1_1006+12del	24.2	27.8	101.0	DS、CS
P10	男	2.5	MPS Ⅱ	智力落后、粗糙面容、关节僵硬、心脏累及	-	?-	21.1	26.9	73.6	DS、HS
P11	男	6.0	MPSⅡ +HSCT	智力倒退、骨骼畸形	IDS	c.121-123delCTC, p.L41del	6.8	1.8	15.7	CS
P12	男	4.7	MPSⅡ +HSCT	智力落后，肝大改善	IDS	c.121-123delCTC, p.L41del	10.3	2.0	21.2	DS、CS
P13	男	4.0	MPSⅡ +HSCT	粗糙面容，心脏累及、精细动作改善、肝大改善	-	?-	13.0	4.9	13.6	DS、CS
P14	男	5.5	MPSⅡ +HSCT	粗糙面容、智力落后、心脏累及，关节僵硬较前改善	-	?-	2.2	3.2	7.6	DS、CS
P15	女	6.3	MPS ⅢA	发育倒退、攻击行为、浓眉、头发粗糙、肝大	SGSH	c.1225del, p.Q409Rfs*4/ c.290_292del, p.H97 _F98delinsL	3.2	14.7	353.3	CS、HS
P16	女	5.9	MPS ⅢA	发育落后、多动、攻击行为、头发粗糙、前额突出、肝大	SGSH	c.959C>T, p.P320L/ c.1297C>T, p.R433W	10.8	2.6	133.0	CS、HS
P17	男	5.6	MPS ⅢB	发育落后、多毛、攻击行为、肝大	NAGLU	c.599C>T, p.A200V/ c.1597C>T,p.R533*	7.7	0.4	69.3	CS、HS
P18	女	5.0	MPS ⅢB	发育落后，粗糙面容、肝大	NAGLU	c.1693C>T, p.R565W/ c.2020C>T, p.R674C	7.6	1.6	33.3	CS、HS
P19	女	1.8	MPS Ⅵ	面容轻微粗陋、关节轻微僵硬、角膜混浊、脊柱后突	ARSB	c.979C>T, p.R327*/ c.1072G>T, p.V358L	82.8	53.5	1.3	CS、DS
P20	男	3.7	MPS Ⅵ	粗糙面容、关节僵硬、角膜混浊	ARSB	c.765T>A, p.Y255/ c.765T>A, p.Y255	173.4	50.6	2.8	DS

表1

图1

表2

表3

表4

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黏多糖	低值混合尿液		高值混合尿液		低值混合尿液		高值混合尿液
黏多糖	均值/mg·L^-1	批内精密度/%	均值/mg·L^-1	批内精密度/%	均值/mg·L^-1	批间精密度/%	均值/mg·L^-1	批间精密度/%
CS	4.35	2.8	48.01	1.9	4.54	2.9	48.23	2.6
HS	5.90	10.4	50.70	5.1	6.10	9.8	53.83	5.1
DS	6.39	6.7	48.33	4.3	6.56	5.7	46.82	4.3

黏多糖	加标前样本浓度/mg·L^-1	理论加标浓度/mg·L^-1	加标后样本浓度/mg·L^-1	平均回收率/%	偏差/%
CS	2.16	2.5	4.3	85.6	1.8
CS	24.25	25	47.98	94.9	1.6
HS	0.44	5	5.96	110.4	12.6
HS	2.95	50	48.72	91.5	2.9
DS	1.83	5	6.67	96.8	3.4
DS	1.66	50	49.94	96.6	3.3

样本	CS		HS		DS
样本	低值	高值	低值	高值	低值	高值
1	97.6	94.5	86.4	86.8	107.7	94.1
2	104.9	95.3	86.6	96.3	106.2	102.5
3	103.6	99.8	86.0	96.2	106.5	86.2
4	98.0	105.2	85.5	104.0	85.7	91.8
5	95.7	108.9	101.3	105.0	109.4	94.1
6	115.5	112.3	96.0	106.0	84.9	86.4