Fauler Guenter, Rechberger Gerald N, Devrnja Danijela, Erwa Wolfgang, Plecko Barbara, Kotanko Peter, Breunig Frank, Paschke Eduard
Department of Pediatrics, Medical University of Graz, Auenbruggerplatz 30, A-8036 Graz, Austria.
Rapid Commun Mass Spectrom. 2005;19(11):1499-506. doi: 10.1002/rcm.1948.
Globotriaosylceramide is a neutral glycolipid containing the trihexoside Gal(alpha1-4)Gal(ss1-4)Glc(ss1-1') covalently bound to N-acylsphingosine. It was identified as the main storage substance in the kidney of patients with Fabry disease, an X-linked deficiency of lysosomal alpha-galactosidase A which can significantly be ameliorated by enzyme replacement therapy. Unlike hemizygote males, affected heterozygote females cannot be identified by enzyme assays and therefore may remain untreated. A quantitation of urinary globotriaosylceramides was proposed as an alternative method for their diagnosis. However, the required studies on physiological and pathological variations in the excretion of trihexosides so far have been prevented by a lack of suitable methods. A validated, robust and quick high-throughput method for the quantitative analysis of globotriaosylceramide isoforms using stable-isotope-dilution/internal standardization and electrospray ionization mass spectrometry (ESI-MS) was developed. An internal standard, stearoyl-d35-globotriaosylceramide, was synthesized by enzymatic coupling of d35-stearic acid to the corresponding lyso-ceramidetrihexoside. Glycolipid isoforms of high purity were obtained from a 5-mL urine portion by extraction on C18 solid-phase columns and a novel washing protocol. ESI-MS analysis was performed in full and neutral loss scan modes. Urinary trihexosyl- and some of the di- and monohexosylceramide isoforms can be quantified within a single experiment. All glycolipid isoforms were above detection limit in healthy male and female subjects (n = 63). Prominent elevations of tetracosanoyl-(C24:0 plus C24:1)-globotriaosylceramides were found in urines from female (>2.5-fold above normals) or male Fabry patients (>5.8-fold above normals), but not among controls. Globotriaosylceramide isoforms shall now be analyzed in Fabry patients, non-genetic kidney disease and healthy subjects to define the conditions for a safe diagnosis of heterozygotes.
球三糖神经酰胺是一种中性糖脂，含有与N-酰基鞘氨醇共价结合的三己糖Gal(α1-4)Gal(β1-4)Glc(β1-1')。它被确定为法布里病患者肾脏中的主要储存物质，法布里病是一种X连锁的溶酶体α-半乳糖苷酶A缺乏症，酶替代疗法可显著改善该病。与半合子男性不同，受影响的杂合子女性无法通过酶测定来识别，因此可能得不到治疗。有人提出对尿中球三糖神经酰胺进行定量分析作为其诊断的替代方法。然而，由于缺乏合适的方法，迄今为止关于三己糖排泄的生理和病理变化所需的研究受到了阻碍。我们开发了一种经过验证的、稳健且快速的高通量方法，用于使用稳定同位素稀释/内标法和电喷雾电离质谱（ESI-MS）对球三糖神经酰胺异构体进行定量分析。通过将d35-硬脂酸与相应的溶血神经酰胺三己糖苷进行酶促偶联，合成了内标硬脂酰-d35-球三糖神经酰胺。通过在C18固相柱上萃取和一种新颖的洗涤方案，从5 mL尿液样本中获得了高纯度的糖脂异构体。ESI-MS分析在全扫描和中性丢失扫描模式下进行。尿中的三己糖基以及一些二己糖基和单己糖基神经酰胺异构体可以在单个实验中进行定量。所有糖脂异构体在健康男性和女性受试者（n = 63）中均高于检测限。在女性（比正常水平高2.5倍以上）或男性法布里病患者（比正常水平高5.8倍以上）的尿液中发现了二十四烷酰基-（C24:0加C24:1）-球三糖神经酰胺显著升高，但在对照组中未发现。现在应在法布里病患者、非遗传性肾病患者和健康受试者中分析球三糖神经酰胺异构体，以确定安全诊断杂合子的条件。
