Fabry disease biomarkers in patients switched from enzyme-replacement therapy to migalastat oral chaperone therapy.

Background: A biomarker profile was evaluated longitudinally in patients with Fabry disease switched from enzyme-replacement therapy (ERT) to migalastat. Methods: 16 Gb3 isoforms and eight lyso-Gb3 analogues were analyzed in plasma and urine by LC-MS/MS at baseline and at three different time points in naive participants and participants switching from either agalsidase α or ß to migalastat. Results: 29 adult participants were recruited internationally (seven centers). The Mainz Severity Score Index and mean biomarker levels remained stable (p ≥ 0.05) over a minimum of 12 months compared with baseline following the treatment switch. Conclusion: In this cohort of patients with Fabry disease with amenable mutations, in the short term, a switch from ERT to migalastat did not have a marked effect on the average biomarker profile.

Methylmalonic acid analysis using urine filter paper samples to screen for metabolic vitamin B12 deficiency in older adults.

Aim: Methylmalonic acid (MMA) analysis in urine represents a noninvasive approach to screening for vitamin B12 deficiency in older adults. A method allowing the analysis of MMA/creatinine in fasting urine collected on filter paper was developed/validated. Method: Dry urine specimens were eluted using a solution containing internal standards, filtrated and analyzed by ultra-performance LC-MS/MS. Results: The method allowed the chromatographic separation of MMA from succinic acid. Dried urine samples were stable for 86 days at room temperature. The MMA/creatinine ratios measured in urine collected on filter paper were highly correlated with values derived from the corresponding liquid specimens. Conclusion: This robust filter paper method might greatly improve the accessibility and cost-effectiveness of vitamin B12 deficiency screening in older adults.

Lysosphingolipid urine screening test using mass spectrometry for the early detection of lysosomal storage disorders.

Background: Sphingolipidoses are caused by a defective sphingolipid catabolism, leading to an accumulation of several glycolipid species in tissues and resulting in neurotoxicity and severe systemic manifestations. Methods & results: Urine samples from controls and patients were purified by solid-phase extraction prior to the analysis by ultra-high-performance liquid chromatography (UPLC) combined with MS/MS. A UPLC-MS/MS method for the analysis of 21 urinary creatinine-normalized biomarkers for eight diseases was developed and validated. Conclusion: Considering the growing demand to identify patients with different sphingolipidoses early and reliably, this methodology will be applied for high-risk screening to target efficiently patients with various sphingolipidoses.

Mass spectrometry analysis of urinary methylmalonic acid to screen for metabolic vitamin B12 deficiency in older adults.

Aim: Vitamin B12 deficiency is characterized metabolically by increased serum and urine methylmalonic acid (MMA). Urinary MMA/creatinine ratio is suggested for screening for metabolic vitamin B12 deficiency in older populations. Results: A UPLC-MS/MS method for the analysis of urinary MMA and creatinine was developed/validated. A good separation of MMA from succinic acid, its structural isomer, was achieved. Intra- and interday accuracy biases and precision coefficients were all ≤6.3% for MMA and creatinine. Urine and serum samples of 34 individuals of the NuAge Biobank were analyzed for technical comparisons showing that urinary MMA/creatinine ratios by UPLC-MS/MS strongly correlated with GC-MS values, and with serum MMA values. Conclusion: The UPLC-MS/MS method developed is rapid/reliable for the analysis of urinary MMA/creatinine ratios.

Tandem mass spectrometry analysis of urinary podocalyxin and podocin in the investigation of podocyturia in women with preeclampsia and Fabry disease patients.

BACKGROUND: Podocytes are highly differentiated visceral cells, and several related specific proteins, such as podocalyxin and podocin are potential tools for the evaluation of podocyturia. However, precise quantitation of podocyturia-related proteins is complex and often unreliable. METHOD: A reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry method was developed and validated to quantify podocalyxin and podocin levels in urine supernatant by using specific cleavable peptides and standards. Urine samples from women with normotensive or hypertensive pregnancies, gestational diabetes and preeclampsia, as well as treated and untreated Fabry patients, and gender-matched controls were investigated. RESULTS: The multiplex analysis shows that podocalyxin levels were higher than podocin levels in patients, the former being particularly higher in pregnant women. Women with preeclampsia had abnormal urine levels of both proteins with a higher sensitivity for podocalyxin. Slightly increased levels of podocin were also observed in Fabry males, while both proteins were increased in untreated Fabry females. Correlations were established between podocalyxin and podocin levels and clinical parameters associated with Fabry disease and preeclampsia. CONCLUSIONS: This methodology makes possible the precise, simultaneous and reliable analysis of podocalyxin and podocin levels, and offers a valuable tool for the evaluation of podocyturia.

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

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 (Ga2), globotriaosylceramide (Gb3), and globotriaosylsphingosine (lyso-Gb3) 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 Ga2 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 Ga2 isoforms/analogues from their lactosylceramide counterparts, was developed and validated in urine. The method was multiplexed with the analysis of 12 Gb3 isoforms/analogues having the same fatty acid moieties as those of Ga2 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 Ga2 from LacCer increased the sensitivity of analysis, especially in women. One untreated Fabry female and two treated Fabry females presented abnormal levels of Ga2 but normal levels of Gb3, supporting the importance of analyzing Ga2, in addition to Gb3. 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.