随机尿游离型NMN、MN HPLC-ED检测方法的建立和临床应用

doi:10.3969/j.issn.1673-8640.2021.05.014

摘要/Abstract

摘要：

目的建立检测随机尿游离型甲氧基去甲肾上腺素（NMN）、甲氧基肾上腺素（MN）的高效液相色谱-电化学（HPLC-ED）方法,并探讨其临床应用价值。方法完善样本处理流程及色谱条件,建立检测随机尿游离型NMN、MN的HPLC-ED法。采用HPLC-ED法检测35例嗜铬细胞瘤患者（嗜铬细胞瘤组）、70例原发性高血压患者（高血压组）、78例非嗜铬细胞瘤肾上腺占位患者（肾上腺占位组）和44名体检健康者（正常对照组）的随机尿和血浆游离型NMN、MN浓度,同时检测肌酐（Cr）,计算尿NMN/Cr比值和尿MN/Cr比值。采用受试者工作特征（ROC）曲线评估各项指标诊断嗜铬细胞瘤的效能。结果 HPLC-ED 法检测随机尿游离型NMN、MN的线性范围均为25~2 500 μg/L,检测限[信噪比（R_SN）=3]分别为5.9和11.3 μg/L,平均回收率为92.8%~103.4%。NMN高、低浓度的批内变异系数（CV）分别为3.7%、2.2%,批间CV分别为5.6%、3.3%;MN高、低浓度批内CV分别为4.3%、2.7%,批间CV分别为5.1%、3.4%。嗜铬细胞瘤组随机尿游离型NMN、MN浓度和尿NMN/Cr比值、尿MN/Cr比值明显高于高血压组、肾上腺占位组及正常对照组（P<0.001）,高血压组、肾上腺占位组及正常对照组之间差异均无统计学意义（P>0.05）。ROC曲线分析结果显示,随机尿游离型NMN、随机尿游离型MN、尿NMN/Cr比值、尿MN/Cr比值、血浆游离型NMN、血浆游离型MN诊断嗜铬细胞瘤的曲线下面积（AUC）分别为0.91、0.88、0.96、0.94、0.93和0.88。嗜铬细胞瘤患者术后1周尿MN/Cr比值均降为正常,但有2例患者术后血浆MN浓度高于术前,有1例患者血浆NMN浓度高于术前。结论建立了检测随机尿游离型NMN、MN浓度的HPLC-ED方法。尿NMN/Cr比值和尿MN/Cr比值诊断嗜铬细胞瘤的效能均优于血浆游离型NMN、MN,且在嗜铬细胞瘤的术后评估中也有较好的价值。

关键词: 甲氧基去甲肾上腺素, 甲氧基肾上腺素, 高效液相色谱-电化学法, 嗜铬细胞瘤

Abstract:

Objective To establish a method for spot urine free normetanephrine（NMN） and metanephrine（MN） by high performance liquid chromatography-electrochemical detection（HPLC-ED）,and to investigate the clinical application role. Methods Sample processing and chromatographic conditions were improved,and HPLC-ED for spot urine free NMN and MN was established. HPLC-ED was used to determine NMN and MN in 35 patients with pheochromocytoma,70 patients with primary hypertension,78 patients with adrenal mass but without pheochromocytoma and 44 healthy subjects. Creatinine（Cr）was determined as well. NMN/Cr ratio and MN/Cr ratio were calculated. Receiver operating characteristic（ROC） curve was used to evaluate the efficiency for diagnosing pheochromocytoma. Results The linear range of HPLC-ED for the determinations of NMN and MN was 25-2 500 μg/L. The determination limits [signal-to-noise ratio（R_SN）=3] were 5.9 and 11.3 μg/L,respectively. The average recovery was 92.8%-103.4%. The within-run coefficients of variation（CV） of high-concentration and low-concentration NMN were 3.7% and 2.2%,and the between-run CV of high-concentration and low-concentration NMN were 5.6% and 3.3%,respectively. The within-run CV of high-concentration and low-concentration MN were 4.3% and 2.7%,and the between-run CV of high-concentration and low-concentration MN were 5.1% and 3.4%,respectively. The concentrations of NMN and MN,NMN/Cr ratio and MN/Cr ratio in pheochromocytoma group were higher than those in hypertension group,adrenal space-occupying group and healthy control group（P<0.001）,and there was no statistical significance among hypertension group,adrenal space-occupying group and healthy control group（P>0.05）. ROC curve analysis showed that the area under curve（AUC） in diagnosing pheochromocytoma of urine free NMN,urine free MN,NMN/Cr ratio,MN/Cr ratio,plasma free NMN and plasma free NMN were 0.91,0.88,0.96,0.94,0.93 and 0.88,respectively. MN/Cr ratio in urine decreased to normal in 1 week after operation in pheochromocytoma patients,but the concentration of plasma MN in 2 patients was higher than that before operation,and in 1 patient,the plasma NMN concentration was higher than that before operation. Conclusions HPLC-ED has been successfully established to determine the concentrations of urine free NMN and MN. Compared with plasma free NMN and MN,spot urine free NMN and MN show good efficiency for the diagnosis of pheochromocytoma,which perform well to evaluate the prognosis of pheochromocytoma.

Key words: Normetanephrine, Metanephrine, High performance liquid chromatography-electrochemical detection, Pheochromocytoma

中图分类号:

R446.1

张磊, 左明, 邹文毕, 王浩然, 张晓清, 丁敏. 随机尿游离型NMN、MN HPLC-ED检测方法的建立和临床应用[J]. 检验医学, 2021, 36(5): 524-529.

ZHANG Lei, ZUO Ming, ZOU Wenbi, WANG Haoran, ZHANG Xiaoqing, DING Min. Method establishment of spot urine free NMN and MN by HPLC-ED and its clinical application[J]. Laboratory Medicine, 2021, 36(5): 524-529.

图/表 7

参考文献 10

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待测物/（μg/L）	回收率/%	CV/%
NMN
25	92.8±1.4	1.5
625	97.4±4.5	4.6
1 250	103.0±4.0	3.9
MN
25	96.9±3.1	3.2
625	95.4±1.0	1.0
1 250	103.4±5.7	5.5

待测物/（μg/L）	回收率/%	CV/%
NMN
25	92.8±1.4	1.5
625	97.4±4.5	4.6
1 250	103.0±4.0	3.9
MN
25	96.9±3.1	3.2
625	95.4±1.0	1.0
1 250	103.4±5.7	5.5

组别	例数	尿NMN/（μg/L）		尿MN/（μg/L）		尿NMN/Cr比值
嗜铬细胞瘤组	35	627.6（307.3~1 129.3）^*#△		239.0（68.6~1 772.1）^*#△		512.7（282.7~865.6）^*#△
高血压组	70	38.3（26.6~75.4）		30.8（17.6~49.9）		32.3（22.1~55.0）
肾上腺占位组	78	29.4（21.5~54.5）		30.6（17.6~48.6）		25.9（17.6~51.8）
正常对照组	44	25.0（14.7~36.3）		29.1（18.1~39.7）		25.5（18.4~36.6）
组别	尿MN/Cr比值/（μg/g Cr）		血浆NMN/（ng/L）		血浆MN/（ng/L）
嗜铬细胞瘤组	176.2（107.1~1 421.0）^*#△		2 005.1（1463.1~4 174.5）^*#△		424.2（136.8~2 223.7）^*#△
高血压组	23.2（16.3~38.5）		93.0（69.6~143.1）		93.0（69.6~143.1）
肾上腺占位组	27.3（17.2~37.4）		79.4（60.2~116.2）		86.4（67.8~119.7）
正常对照组	26.9（21.9~42.6）		73.1（50.6~98.9）		45.8（29.9~72.2）

组别	例数	尿NMN/（μg/L）		尿MN/（μg/L）		尿NMN/Cr比值
嗜铬细胞瘤组	35	627.6（307.3~1 129.3）^*#△		239.0（68.6~1 772.1）^*#△		512.7（282.7~865.6）^*#△
高血压组	70	38.3（26.6~75.4）		30.8（17.6~49.9）		32.3（22.1~55.0）
肾上腺占位组	78	29.4（21.5~54.5）		30.6（17.6~48.6）		25.9（17.6~51.8）
正常对照组	44	25.0（14.7~36.3）		29.1（18.1~39.7）		25.5（18.4~36.6）
组别	尿MN/Cr比值/（μg/g Cr）		血浆NMN/（ng/L）		血浆MN/（ng/L）
嗜铬细胞瘤组	176.2（107.1~1 421.0）^*#△		2 005.1（1463.1~4 174.5）^*#△		424.2（136.8~2 223.7）^*#△
高血压组	23.2（16.3~38.5）		93.0（69.6~143.1）		93.0（69.6~143.1）
肾上腺占位组	27.3（17.2~37.4）		79.4（60.2~116.2）		86.4（67.8~119.7）
正常对照组	26.9（21.9~42.6）		73.1（50.6~98.9）		45.8（29.9~72.2）

项目	AUC（95%可信区间）	最佳临界值	敏感性/%	特异性/%	Youden指数
随机尿
NMN	0.90（0.83~0.98）	153.6 μg/L	77.1	95.5	0.726
MN	0.87（0.79~0.95）	47.5 μg/L	85.7	79.9	0.656
NMN/Cr比值	0.95（0.90~1.00）	122.5 μg/g Cr	85.7	98.3	0.840
MN/Cr比值	0.94（0.89~0.99）	65.0 μg/g Cr	85.7	95.0	0.807
血浆
NMN	0.93（0.87~0.98）	431.4 ng/L	82.9	92.7	0.756
MN	0.88（0.82~0.93）	131.2 ng/L	80.0	84.4	0.644