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

Abstract

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the α-galactosidase A enzyme. This enzyme cleaves the last sugar unit of glycosphingolipids, including globotriaosylceramide (Gb₃), globotriaosylsphingosine (lyso-Gb₃), and galabiosylceramide (Ga₂). Enzyme impairment leads to substrate accumulation in different organs, vascular endothelia, and biological fluids. Enzyme replacement therapy (ERT) is a commonly used treatment. Urinary analysis of Gb₃ isoforms (different fatty acid moieties), as well as lyso-Gb₃ and its analogues, is a reliable way to monitor treatment. These analogues correspond to lyso-Gb₃ with chemical modifications on the sphingosine moiety (-C₂H₄, -C₂H₄+O, -H₂, -H₂+O, +O, +H₂O₂, and +H₂O₃). The effects of sample collection time on urinary biomarker levels between ERT cycles were not previously documented. The main objective of this project was to analyze the aforementioned biomarkers in urine samples from seven Fabry disease patients (three treated males, three treated females, and one ERT-naïve male) collected twice a day (morning and evening) for 42 days (three ERT cycles). Except for one participant, our results show that the biomarker levels were generally more elevated in the evening. However, there was less variability in samples collected in the morning. No cyclic variations in biomarker levels were observed between ERT infusions.

Keywords: Fabry disease; diurnal variation; globotriaosylceramide; globotriaosylsphingosine; glycosphingolipids; mass spectrometry.

Figure 1

Mean concentration of Fabry biomarkers for three enzyme replacement therapy (ERT) cycles (n = 42 days) at two different collection times in seven study participants. All participants were under ERT, except participant no. 1 who never received treatment. Patients 1–4 were males, whereas patients 5–7 were females. For lyso-Gb₃ analogues, the mass difference in Da compared to lyso-Gb₃ is indicated in brackets. ** corrected p-value ≤ 0.001 for the paired-sample t-test; † corrected p-value ≤ 0.001 for Levene’s test for equality of variances.

Figure 2

Longitudinal variation (n = 42) of Fabry biomarker levels in a 30-year-old untreated male (no ERT) (participant no. 1). For lyso-Gb₃ analogues, the mass difference in Da compared to lyso-Gb₃ is indicated in brackets.

Figure 3

Longitudinal variation (n = 42) of Fabry disease biomarker levels during three ERT cycles in male patients. Results are shown for morning urine collection only. For lyso-Gb₃ analogues, the mass difference in Da compared to lyso-Gb₃ is indicated in brackets. Vertical lines indicate the time points where Fabry patients received their ERT infusions.

Figure 4

Longitudinal variation (n = 42) of Fabry disease biomarker levels during three ERT cycles in female patients. Results are shown for morning urine collection only. The vertical lines indicate the time points where Fabry patients received their ERT infusions. For lyso-Gb₃ analogues, the mass difference in Da compared to lyso-Gb₃ is indicated in brackets.