Clifford-Mobley Oliver, Hewitt Laura, Rumsby Gill
Department of Clinical Biochemistry, University College London Hospitals NHS Foundation Trust, London, UK
Department of Clinical Biochemistry, University College London Hospitals NHS Foundation Trust, London, UK.
Ann Clin Biochem. 2016 Jul;53(Pt 4):485-94. doi: 10.1177/0004563215606158. Epub 2015 Sep 4.
The primary hyperoxalurias are inherited disorders of glyoxylate metabolism, which cause over-production of oxalate leading to urolithiasis and subsequent renal failure. Other metabolites may be produced in excess in the different forms of PH: glycolate in PH1, glycerate in PH2 and 4-hydroxy-2-oxoglutarate and 2,4-dihydroxyglutarate in PH3. The aim of this study was to set up and validate a method for the simultaneous analysis of these metabolites in urine and to evaluate its use for preliminary identification of primary hyperoxaluria prior to definitive diagnosis by genetic testing.
Urine samples were derivitized by methoximation and silylation and extracted into organic solvent prior to analysis by gas chromatography mass spectrometry.
Recovery of the analytes spiked into urine ranged from 91 to 103% and total analytical imprecision ranged from 3.0 to 13.6%. 4-Hydroxy-2-oxoglutarate was unstable in urine at room temperature, and preservation by acidification was required. Mean urinary glycolate, glycerate and 4-hydroxy-2-oxoglutarate or 2,4-dihydroxyglutarate (expressed as a ratio to creatinine) were significantly higher in patients with PH1, PH2 and PH3, respectively. Low 4-hydroxy-2-oxoglutarate was observed in some patients with PH3, probably due to the instability of this analyte, but all PH3 patients had elevated 2,4-dihydroxyglutarate. During five months of routine service, seven cases of PH were identified by this method and subsequently confirmed by gene sequencing including two with novel mutations in HOGA1.
This study confirms that the method is useful in aiding the diagnosis of primary hyperoxaluria and can direct genetic testing.
原发性高草酸尿症是乙醛酸代谢的遗传性疾病，可导致草酸盐过度生成，进而引发尿石症及随后的肾衰竭。在不同类型的原发性高草酸尿症（PH）中，可能会过量产生其他代谢产物：PH1型中为乙醇酸，PH2型中为甘油酸，PH3型中为4-羟基-2-氧代戊二酸和2,4-二羟基戊二酸。本研究的目的是建立并验证一种同时分析尿液中这些代谢产物的方法，并评估其在通过基因检测进行明确诊断之前用于原发性高草酸尿症初步鉴定的效用。
尿液样本经甲氧胺化和硅烷化衍生化处理，然后萃取到有机溶剂中，再通过气相色谱 - 质谱联用仪进行分析。
添加到尿液中的分析物回收率在91%至103%之间，总分析不精密度在3.0%至13.6%之间。4-羟基-2-氧代戊二酸在室温下于尿液中不稳定，需要通过酸化进行保存。PH1、PH2和PH3患者尿液中乙醇酸、甘油酸以及4-羟基-2-氧代戊二酸或2,4-二羟基戊二酸（以与肌酐的比值表示）的平均水平分别显著升高。在一些PH3患者中观察到4-羟基-2-氧代戊二酸水平较低，可能是由于该分析物的不稳定性，但所有PH3患者的2,4-二羟基戊二酸水平均升高。在五个月的常规检测中，通过该方法鉴定出7例原发性高草酸尿症病例，随后经基因测序得到证实，其中包括2例HOGA1基因有新突变的病例。
本研究证实该方法有助于原发性高草酸尿症的诊断，并可指导基因检测。
