Background

The prognosis of patients with Hereditary Tyrosinemia Type 1 (HT-1) has greatly improved with early detection through newborn screening and the introduction of nitisinone (NTBC) therapy. A recent guideline calls for periodic monitoring of biochemical markers and NTBC levels to tailor treatment; however, this is currently only achieved through a combination of clinical laboratory tests. We developed a multiplexed assay measuring relevant amino acids, succinylacetone (SUAC), and NTBC in dried blood spots (DBS) to facilitate treatment monitoring.

Methods

Tyrosine, phenylalanine, methionine, NTBC and SUAC were eluted from DBS with methanol containing internal standards for each analyte and analyzed by liquid chromatography tandem mass spectrometry over 6.5 min in the multiple reaction monitoring positive mode.

Results

Pre-analytical and analytical factors were studied and demonstrated a reliable assay. Chromatography resolved an unknown substance that falsely elevates SUAC concentrations and was present in all samples. To establish control and disease ranges, the method was applied to DBS collected from controls (n = 284) and affected patients before (n = 2) and after initiation of treatment (n = 29). In the treated patients SUAC concentrations were within the normal range over a wide range of NTBC levels.

Conclusions

This assay enables combined, accurate measurement of revelevant metabolites and NTBC in order to simplify treatment monitoring of patients with HT-1. In addition, the use of DBS allows for specimen collection at home to facilitate more standardization in relation to drug and dietary treatment.

中文翻译：

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实验室监测的I型遗传性酪氨酸血症患者。

背景

通过新生儿筛查和引入尼替尼酮（NTBC）治疗，可以及早发现，从而大大改善了1型遗传性酪氨酸血症（HT-1）患者的预后。最近的指南要求定期监测生化标志物和NTBC水平以调整治疗；但是，目前只能通过结合临床实验室测试来实现。我们开发了一种用于测定干血斑（DBS）中相关氨基酸，琥珀酰丙酮（SUAC）和NTBC的多元测定法，以促进治疗监测。

方法

酪氨酸，苯丙氨酸，蛋氨酸，NTBC和SUAC用含有内标物的甲醇从DBS中洗脱出来，用于每种分析物，并通过液相色谱串联质谱法在6.5分钟内以多重反应监测阳性模式进行分析。

结果

对分析前和分析因素进行了研究，并证明了该方法的可靠性。色谱分离出一种未知物质，该物质错误地提高了SUAC浓度，并存在于所有样品中。为了建立控制范围和疾病范围，该方法适用于从对照组（n  = 284）和受影响患者在治疗开始之前（n = 2）和开始治疗后（n  = 29）收集的DBS 。在接受治疗的患者中，SUAC的浓度在大范围的NTBC水平内都在正常范围内。

结论

该测定法能够准确，准确地测量后代代谢物和NTBC，以简化对HT-1患者的治疗监测。此外，使用DBS可以在家中采集标本，以促进与药物和饮食治疗有关的更多标准化。