Novel Aspect: Detection and quantitation of psychosine in human urine using tandem mass spectrometry.

Introduction:

Psychosine (galactosylsphingosine, m/z 462.2) is a sphingolipid intermediate formed via a side chain reaction from galactosylceramide. Galactocerebroside b -galactosidase (GALC) is the enzyme responsible for the catabolism of galactosylceramide to ceramide and galactose. Those affected with Krabbe's disease (Globoid Cell Leukodystrophy) have a defect in this enzyme causing galactosylceramide to be converted into psychosine. Like other sphingolipodoses, Krabbe's disease can also arise from a deficiency of the activators saposin A and/or C (1). Tissue accumulation of psychosine leads to demyelination of CNS and PNS axons and characteristic aggregation of multinucleated globoid cells (2). Current methods of diagnosis include GALC enzyme assays using skin fibroblasts or blood, DNA testing and imaging studies.

Methods and Instrumentation:

Extraction procedure: Urine (500 m L) was dried in a vacuum concentrator. CHCl₃/MeOH (5/1, 500 μL) was added and the samples were vortexed and sonicated in a warm bath to dislodge the solids. The samples were again vortexed and centrifuged. The organic phase was removed, dried under a N₂ stream and re-dissolved in 500 μL CHCl₃/MeOH (5/1). For analysis, 25 μL of sample was added to 5 μL of internal standard solution (N-acetyl psychosine, 30 pmol/μL in CHCl₃/MeOH, 5/1).

ESI-MS/MS-MRM: The mass spectrometer (PE SCIEX API III⁺ triple quadrupole mass spectrometer) was tuned and calibrated in the positive ion mode using settings in which the carbon-13 isotope ions from the PPG calibrant were not resolved from one another. Flow injection analysis (FIA) was done using 4:4:1 CHCl₃/MeOH/0.1% HCOOH at a flow rate of 35 μL/min with 15 μL sample injections. Under these conditions psychosine gives a parent ion at m/z of 462.2 with CAD fragment ions at m/z 282 and 264 corresponding to dehydrated galactosyl moieties, while N-acetyl psychosine yields a parent ion at m/z 504 and similar CAD fragment ions. MS/MS-MRM used four transitions: m/z 462-->282 (psychosine), 462-->264 (psychosine), and 504-->264 (internal standard). Quantitative measurements were made by integration of the responses from psychosine and the internal standard.

Preliminary Data:

Preliminary experiments showed optimal fragment ion spectra and multiple reaction monitoring (MRM) conditions were obtained with an orifice voltage of 100, a collision gas thickness instrument setting of 125 and a rod offset (R0-R2) of 10V. Quantitative measurements were obtained by taking the peak areas of the psychosine transitions and normalizing them to the peak areas of the internal standard transition. Preliminary data suggest that this assay is sufficiently sensitive to measure psychosine excretion in control urine samples, and future work will focus on urine from Krabbe's patients to establish the possible clinical utility of this assay.

References:

1. Harzer, Klaus; Hiraiwa, Masao; Paton Barbara C. Saposins (sap) A and C Activate the Degradation of Galactosylsphingosine. FEBS Letters (2001), 508 (1), 107-110.

2. Cotran, Ramzi. S.; Kumar, Vinay; Collins, Tucker (1999). Robbins Pathologic Basis of Disease, 6 th Edition, p. 1339, Saunders , New York.