Zhang Haoyue, Wood Tim, Young Sarah P, Millington David S
Duke Medicine, Pediatrics, Medical Genetics Division, NC, USA.
Greenwood Genetic Center, SC, USA.
Mol Genet Metab. 2015 Feb;114(2):123-8. doi: 10.1016/j.ymgme.2014.09.009. Epub 2014 Oct 5.
Mucopolysaccharidoses (MPS) are complex storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, brain and other tissues. Symptomatic patients are typically screened for MPS by analysis of GAG in urine. Current screening methods used in clinical laboratories are based on colorimetric assays that lack the sensitivity and specificity to reliably detect mild GAG elevations that occur in some patients with MPS. We have developed a straightforward, reliable method to quantify chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) in urine by stable isotope dilution tandem mass spectrometry. The GAGs were methanolyzed to uronic acid-N-acetylhexosamine or iduronic acid-N-glucosamine dimers and mixed with stable isotope labeled internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS and CS were separated by ultra-performance liquid chromatography and analyzed by electrospray ionization tandem mass spectrometry using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. The method was robust with a mean inaccuracy from 1 to 15%, imprecision below 11%, and a lower limit of quantification of 0.4mg/L for CS, DS and HS. We demonstrate that the method has the required sensitivity and specificity to discriminate patients with MPS III, MPS IVA and MPS VI from those with MPS I or MPS II and can detect mildly elevated GAG species relative to age-specific reference intervals. This assay may also be used for the monitoring of patients following therapeutic intervention. Patients with MPS IVB are, however, not detectable by this method.
黏多糖贮积症（MPS）是复杂的贮积性疾病，可导致糖胺聚糖（GAGs）在尿液、血液、大脑和其他组织中蓄积。有症状的患者通常通过分析尿液中的GAGs来筛查MPS。临床实验室目前使用的筛查方法基于比色测定法，缺乏可靠检测某些MPS患者中出现的轻度GAG升高的灵敏度和特异性。我们开发了一种简单可靠的方法，通过稳定同位素稀释串联质谱法定量尿液中的硫酸软骨素（CS）、硫酸皮肤素（DS）和硫酸乙酰肝素（HS）。将GAGs进行甲醇解生成糖醛酸-N-乙酰己糖胺或艾杜糖醛酸-N-葡糖胺二聚体，并与由GAG标准品的氘代甲醇解衍生而来的稳定同位素标记内标混合。通过超高效液相色谱法分离HS、DS和CS衍生的特定二聚体，并使用针对每种目标GAG产物及其相应内标的选择反应监测，通过电喷雾电离串联质谱法进行分析。该方法稳健，平均误差为1%至15%，不精密度低于11%，CS、DS和HS的定量下限为0.4mg/L。我们证明该方法具有区分MPS III、MPS IVA和MPS VI患者与MPS I或MPS II患者所需的灵敏度和特异性，并且相对于年龄特异性参考区间可以检测到轻度升高的GAG种类。该检测方法也可用于治疗干预后患者的监测。然而，该方法无法检测出MPS IVB患者。
