Separation and Analysis of Lactosylceramide, Galabiosylceramide, and Globotriaosylceramide by LC-MS/MS in Urine of Fabry Disease Patients

Abstract

Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A (α-GAL A) deficiency. This enzyme contributes to the cellular recycling of glycosphingolipids such as galabiosylceramide (Ga), globotriaosylceramide (Gb), and globotriaosylsphingosine (lyso-Gb) by hydrolyzing the terminal α-galactosyl moiety. Urine and plasma α-GAL A substrates are currently analyzed as biomarkers for the detection, monitoring, and follow-up of Fabry disease patients. The sensitivity of the analysis of Ga is decreased by the co-analysis of its structural isomer, lactosylceramide (LacCer), which is not an α-GAL A substrate. A normal-phase ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology, allowing the baseline separation of 12 Ga isoforms/analogues from their lactosylceramide counterparts, was developed and validated in urine. The method was multiplexed with the analysis of 12 Gb isoforms/analogues having the same fatty acid moieties as those of Ga for comparison, and with creatinine for sample normalization. Urine samples were studied from 34 untreated and 33 Fabry males treated by enzyme replacement therapy (ERT) and 54 untreated and 19 ERT-treated Fabry females, along with 34 male and 25 female healthy controls. The chromatographic separation of Ga from LacCer increased the sensitivity of analysis, especially in women. One untreated Fabry female and two treated Fabry females presented abnormal levels of Ga but normal levels of Gb, supporting the importance of analyzing Ga in addition to Gb. Our results show that urine LacCer levels from females were significantly higher than those from males. Moreover, LacCer levels were not affected by Fabry disease for both males and females.

Effects of Enzyme Replacement Therapy and Antidrug Antibodies in Patients with Fabry Disease

“…3 The deacetylated and, therefore, soluble form of Gb3, lyso-Gb3, seems to be a reliable biomarker for both disease progression and therapy efficacy, and can be detected in blood plasma as well as urine. [4][5][6][7][8][9][10] The systemic cellular accumulation of Gb3 leads to progressive renal failure, cardiomyopathy, and recurrent cerebrovascular events, significantly limiting life expectancy in affected patients. 3 The most common cause of death is cardiovascular disease.…”

Altered Sphingolipids Metabolism Damaged Mitochondrial Functions: Lessons Learned From Gaucher and Fabry Diseases

“…α-Gal A catalyzes the lysosomal hydrolysis of globotriaosylceramide (Gb-3) to lactosylceramide and digalactosylceramide (Gal-Gal-Cer) to galactosylceramide (Gal-Cer) [57,58]. The deficiency of this enzyme leads to an accumulation of Gb-3, its metabolite, globotriaosylsphingosine (Lyso-Gb-3), and Gal-Gal-Cer in lysosomes (Figure 1) [59]. Detection of Gb-3, Lyso-Gb-3, and Gal-Gal-Cer in urine and plasma are the standard diagnostic methods for FD [58].…”

Diurnal Variation of Urinary Fabry Disease Biomarkers during Enzyme Replacement Therapy Cycles

“…The function of this glycoside hydrolase is to cleave the terminal α-galactosyl moieties from glycoproteins or glycolipids [ 1 ]. The deficiency of the enzyme leads to the accumulation of substrates such as globotriaosylsphingosine (lyso-Gb 3 ), galabiosylceramide (Ga 2 ), and globotriaosylceramide (Gb 3 ) in cells [ 4 ], tissues [ 5 ], and biological fluids [ 6 , 7 , 8 , 9 ]. Different untargeted metabolomic studies performed in our laboratory with urine and plasma specimens from FD patients identified different analogues of lyso-Gb 3 [ 10 , 11 ], as well as analogues/isoforms of Ga 2 [ 12 ] and Gb 3 [ 13 ], as FD biomarkers.…”