The Role of Chromatography in the Separation of Glucosylceramide Isoforms from Their Isobaric Galactosylceramide Counterparts in the Era of Precision Medicine Christiane Auray-Blais (Presenter) Universite de Sherbrooke Presenter Bio: Christiane Auray-Blais is the Director of the Neonatal Urine Screening Program for hereditary metabolic disorders in Sherbrooke, QC. More than 3 300 000 newborn babies were screened in the Province of Quebec for 25 disorders of amino acids and organic acids. She holds a Ph.D. in radiobiology from the Faculty of Medicine and Health Sciences (FMHS) at the Université de Sherbrooke and postdoctoral studies from Duke University Medical Center in North Carolina, US. She has a master’s degree in Health Law from the Faculty of Law at the Université de Sherbrooke and a bachelor’s degree in biochemistry. She is the author of more than 270 publications, abstracts and articles. She is a professor in the Medical Genetics Division in the Department of Pediatrics at the FMHS and a researcher at the Clinical Research Centre at CHUS in Sherbrooke, and in the Mother-Child Axis.

Authors: Christiane Auray-Blais (1), Michel Boutin (1), Sun Y (2), John Shacka (3)
(1) Universite de Sherbrooke, Sherbrooke, QC, (2) Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, (3) University of Alabama at Birmingham, AL

Glucosylceramide (GluCer) is a glycosphingolipid associated with various diseases, such as Parkinson’s disease and Gaucher disease. It is comprised of different fatty acid chains (isoforms), a sphingosine and a glucose moiety. The only difference between GluCer and galactosylceramide (GalCer) is the conformation of one hydroxyl group: axial in the former and equatorial in the latter. The focus of this study was to separate chromatographically GluCer isoforms from their isobaric GalCer counterparts. We report an ultra-performance liquid chromatography (UPLC) coupled to a tandem mass spectrometry (MS/MS) 6-min methodology which provides an efficient chromatographic separation of GluCer isoforms from their GalCer counterparts in brain tissues of Parkinson’s disease patients. This method was successfully applied to other matrices, such as vitreous humour in a Gaucher disease patient.

Introduction

Glucosylceramide (GluCer), also known as glucocerebroside, is a glycosphingolipid associated with various diseases, such as Parkinson’s disease and Gaucher disease. It is comprised of different fatty acid chains (isoforms), a sphingosine and a glucose moiety. The only difference between GluCer and galactosylceramide (GalCer) is the conformation of one hydroxyl group: axial in the former and equatorial in the latter. Throughout the years, the analysis of GluCer in various biological fluids or other matrices has led to erroneous results because the methodologies or technologies used could not adequately separate both molecules in an efficient and reliable way. It was indeed a challenge to separate GluCer isoforms from their GalCer counterparts since the latter are usually in higher abundance, more specifically in brain tissue matrices. The main objective of our study was thus to separate chromatographically GluCer isoforms from their isobaric GalCer counterparts. As a proof of concept, we report ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS) results of GluCer isoforms clearly separated from their GalCer counterparts in brain tissues of Parkinson’s disease patients (1).

Methods

One hundred (100) mg of frozen brain tissues from Parkinson’s disease patients and controls were homogenized with 400 µL of MeOH at high-speed (5 m/s) for 45 s using a Bead Ruptor (Omni) device with ceramic beads, followed by the addition of 600 µL of MeOH homogenized again for 45 s at low speed (3 m/s). The purification of the homogenates was performed according to an adapted process from Brignol et al.(2) One mL of MeOH, plus 55 ìL of DMSO, and 20 ìL of the GluCer(C16:0)D3 internal standard (at 10 ìmol/L in DMSO) were added to 100 ìL of brain tissue homogenates, followed by the addition of 625 ìL of acetone and 300 ìL of H2O. Samples were then vortexed (30 min), centrifuged (1 min) to recover the supernatant which was diluted in 300 ìL of H2O and purified with a hydrophilic-lipophylic balance (HLB) solid phase extraction cartridge. The eluates were evaporated to dryness under a stream of nitrogen and resuspended with 50 ìL of DMSO and 200 ìL of the mobile phase A (ACN 95%/MeOH 2.5%/H2O 2.5%/FA 0.5%/5 mM Amm. Form.) for UPLC analysis on an Acquity I-Class sytem (Waters Corp.) using a Halo Hilic column (Advanced Materials Technology). The Xevo TQ-S tandem MS system (Waters) working in multiple reaction monitoring (MRM) mode was used. Seven MRM reactions were analyzed: the calibration curve standard GalCer(C15:0) (m/z 686.55), the internal standard GluCer(C16:0)D3 (m/z 703.59), and the following five specific GluCer/GalCer isoforms (C18:0) (m/z 728.60), (C20:0) (m/z 756.64), (C22:0) (m/z 784.67), (C24:1) (m/z 810.68), and (C24:0) (m/z 812.70).

Results

The liquid chromatography method developed allowed the baseline separation of 5 GluCer isoforms from their GalCer isomers. The validation of the method provided acceptable results for GluCer isoforms: LOD varied from 0.4 and 1.1 nmol/g brain, LOQ from 1.2 and 3.7 nmol/g brain, intra- and interday precisions were <16% for GluCer(C15:0) in LQCs and HQCs and for 5 GluCer isoforms under study in the pooled control brain tissues. Intra- and interday accuracies were <24% and <9% for LQCs and HQCs, respectively. No significant adhesion to plastic or glass was observed after three transfers (bias <15%) for both GalCer(C15:0) and GluCer isoforms. A 7-point calibration curve provided a mean correlation coefficient (R2) of 0.994 (n = 5). Recoveries for GluCer(C15:0) at LQCs and HQCs were 32.3 and 25.2%, respectively.

Conclusions & Discussion

We have achieved a good separation between GluCer isoforms and their GalCer counterparts in brain tissues of Parkinson’s disease patients and controls using a normal phase chromatography. A good chromatographic separation was necessary because GalCer isoform levels were found to be up to 300 times higher than GluCer isoforms. The principles of this methodology have been successfully applied to other matrices such as vitreous humour from a Gaucher patient in order to obtain reliable and robust results in an era of precision medicine.

References

1. Boutin M, Sun Y, Shacka JJ, Auray-Blais C., Anal. Chem. 88(3):1856-63, 2016.

2. Brignol N, Chang K, Hamler R, Schilling A.E, Khanna R, Lockhart D.J, Clark S.W, Benjamin E.R. Mol. Genet. Metab. 105 (2), S22, 2012.

Acknowledgements

We would like to thank Waters Corp. for their technical and scientific support. We are also grateful to the Michael J Fox Foundation for their grant support.