Rapid Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry Assay for a Characteristic Glycogen-Derived Tetrasaccharide in Pompe Disease and Other Glycogen Storage Diseases

Abstract

BACKGROUND Urinary excretion of the tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc4) is increased in various clinical conditions associated with increased turnover or storage of glycogen, making Glc4 a potential biomarker for glycogen storage diseases (GSD). We developed an ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay to detect Glc4 in urine without interference of the Glc4 isomer maltotetraose (M4). METHODS Urine samples, diluted in 0.1% ammonium hydroxide containing the internal standard acarbose, were filtered, and the filtrate was analyzed by UPLC-MS/MS. RESULTS We separated and quantified acarbose, M4, and Glc4 using the ion pairs m/z 644/161, 665/161, and 665/179, respectively. Response of Glc4 was linear up to 1500 μmol/L and the limit of quantification was 2.8 μmol/L. Intra- and interassay CVs were 18.0% and 18.4% (10 μmol/L Glc4), and 10.5% and 16.2% (200 μmol/L Glc4). Glc4 in control individuals (n = 116) decreased with increasing age from a mean value of 8.9 mmol/mol to 1.0 mmol/mol creatinine. M4 was present in 5% of urine samples. Mean Glc4 concentrations per age group in untreated patients with Pompe disease (GSD type II) (n = 66) were significantly higher, ranging from 39.4 to 10.3 mmol/mol creatinine (P < 0.001–0.005). The diagnostic sensitivity of Glc4 for GSD-II was 98.5% and the diagnostic specificity 92%. Urine Glc4 was also increased in GSD-III (8 of 9), GSD-IV (2 of 3) and GSD-IX (6 of 10) patients. CONCLUSIONS The UPLC-MS/MS assay of Glc4 in urine was discriminative between Glc4 and M4 and confirmed the diagnosis in >98% of GSD-II cases.

Ultra-performance liquid chromatography/tandem mass spectrometry for determination of sulfatides in dried blood spots from patients with metachromatic leukodystrophy

“…Accumulating metabolites or proteins specifically secreted by storage cells are good candidate biomarkers for monitoring ERT in LSDs. Globotriosylceramide (Gb3) for Fabry disease, lyso‐GM2 ganglioside for Tay‐Sachs disease and Sandhoff disease, and tetrasaccharide 6‐α‐D‐glucopyranosyl‐maltotriose (Glc 4 ) for Pompe disease are good examples . Likewise, sulfatide detection in urine or blood could be used to monitor therapeutic efficacy in MLD.…”

Long‐term complications of glycogen storage disease type Ia in the canine model treated with gene replacement therapy

“…Increased urinary biomarker Glc 4 (Lennartson et al 1976;Oberholzer and Sewell 1990;Sluiter et al 2012) Not described Anemia (Wang et al 2012) Occasionally elevated blood glucose in dog R (unpublished observation)…”

Laboratory studies and Pompe disease: from suspicion to therapy monitoring

“…У боль-шинства пациентов с БП отмечается повышение Glc4 в моче (при младенческой форме более выраженное, чем при БППД). Этот анализ служит дополнительным подтверждением диагноза [31,32]. В России данный анализ не выполняется.…”

“…Отсутствие нормализации Glc4 в течение первых нескольких недель терапии связано с минимальным улучшением моторных функций и плохим прогнозом. Напротив, быстрое снижение уровня Glc4 спустя 4 нед терапии и поддержание такого низкого уровня в дальнейшем ассоциировано с благоприятным прог-нозом [43,44].…”

“…По опуб-ликованным данным, средняя концентрация Glc4 в моче здоровых детей в возрасте до 1 года составляет 8,9 ммоль/моль креатинина (2,4-16,2 ммоль/моль креатинина), а в той же возрастной группе больных с младенческой формой БП -39,4 ммоль/моль креа-тинина (16,6-74,2 ммоль/моль креатинина) [32]. По-казано, что у младенцев с младенческой формой БП, выявленной благодаря неонатальному скринингу, средняя концентрация Glc4 в моче значимо ниже (25 ммоль/моль креатинина (18-32 ммоль/моль креа-тинина), чем у той же группы пациентов, выявленных по наличию симптомов БП (38 ммоль/моль креатинина (24-42 ммоль/моль креатинина) [45].…”