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. 2016 Sep;8(17):1793-807.
doi: 10.4155/bio-2016-0116. Epub 2016 Aug 15.

Relative distribution of Gb3 isoforms/analogs in NOD/SCID/Fabry mice tissues determined by tandem mass spectrometry

Philippe Provençal  1 , Michel Boutin  1 , Shaalee Dworski  2 , Bryan Au  2 , Jeffrey A Medin  2   3 , Christiane Auray-Blais  1
Affiliations

Relative distribution of Gb3 isoforms/analogs in NOD/SCID/Fabry mice tissues determined by tandem mass spectrometry

Philippe Provençal et al. Bioanalysis. 2016 Sep.

Abstract

Aim: Fabry disease is a lysosomal storage disorder leading to glycosphingolipid accumulation in different organs, tissues and biological fluids. The development of a Fabry disease gene therapy trial is underway in Canada. A tool to determine the distribution of Gb3 biomarkers in tissues of Fabry mice might be applicable to monitor the effect of gene therapy. Results & methodology: An ultra-performance LC-MS/MS (UPLC-MS/MS) method for the analysis of 22 Gb3 isoform/analogs in various Fabry mice tissues was developed and validated. Marked variation in biomarker organ distribution was found with higher levels in the spleen, followed by the small intestine, kidneys, lungs, heart, liver and brain.

Conclusion: The devised method is sensitive and useful for the evaluation of biomarker profiles in Fabry mice.

Keywords: Fabry disease; MS; NOD/SCID mice; globotriaosylceramide; isoforms/analogs; liquid–liquid extraction; organ biomarker distribution.

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Conflict of interest statement

Financial & competing interests disclosure This research was funded by a Grant-in-Aid of research from the Canadian Institutes of Health Research (CIHR, 129737). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Figures

<b>Figure 1.</b>
Figure 1.. Chemical structure of globotriaosylceramide biomarkers with the example of Gb3[(d18:1)(C22:0)] from Group 1.
Groups 2–5 show the possible modifications on the sphingosine and/or fatty acid chain. Gb3: Globotriaosylceramide.
<b>Figure 2.</b>
Figure 2.. Overlay ion chromatograms of globotriaosylceramide isoforms/analogs in kidney tissues of an NSF mouse.
IS: Gb3 [(d18:1)(C18:0)D3]. Each chromatogram was zoomed to reveal the biomarker of interest. Gb3 isoforms/analogs are shown in the table according to their retention time appearance on the chromatogram. CPS: Counts per second; Gb3: Globotriaosylceramide; IS: Internal standard.
<b>Figure 3.</b>
Figure 3.. Biomarker profile for globotriaosylceramide isoforms/analogs for different male (n = 9) and female (n = 9) nonobese diabetic/severe combined immune deficiency/Fabry mice organ tissues: brain, liver, heart, lung, kidney, small intestine and spleen; and plasma samples (females: n = 9, males: n = 9).
Results are expressed as the area of each compound/area of the Gb3[(d18:1)(C18:0)D3] IS, which is the response factor. The number before each biomarker refers to: Group 1: Gb3 isoforms with saturated fatty acids; Group 2: Gb3 isoforms/analogs with one extra-double bond (on the sphingosine or fatty acid); Group 3: Gb3 isoforms/analogs with two extra-double bonds (on the sphingosine and fatty acid or both on the fatty acid); Group 4: Gb3 isoforms hydroxylated fatty acid; and Group 5: methylated Gb3 isoforms. *For Gb3 isoforms/analogs with one or two extra-double bonds, the isoforms with the extra-double bond(s) on the fatty acids were analyzed together with their structural isomers with one extra-double bond on the sphingosine moiety (analogs). Error bars correspond to the mean plus one standard deviation. Gb3: Globotriaosylceramide; IS: Internal standard; nd: Not detected.
<b>Figure 4.</b>
Figure 4.. Biomarker profile comparison for globotriaosylceramide isoforms/analogs for brain, heart and spleen of NSF male (n = 9) and female Fabry mice tissues (n = 9).
Results are expressed as the area of each compound/area of the Gb3[(d18:1)(C18:0)D3] IS, which is the response factor. The number before each biomarker refers to: Group 1: Gb3 isoforms with saturated fatty acids; Group 2: Gb3 isoforms/analogs with one extra-double bond (on the sphingosine or fatty acid); Group 3: Gb3 isoforms/analogs with two extra-double bonds (on the sphingosine and fatty acid or both on the fatty acid); Group 4: Gb3 isoforms hydroxylated fatty acid; and Group 5: methylated Gb3 isoforms. *For Gb3 isoforms/analogs with one or two extra-double bonds, the isoforms with the extra-double bond(s) on the fatty acids were analyzed together with their structural isomers with one extra-double bond on the sphingosine moiety (analogs). Error bars correspond to the mean plus one standard deviation. Gb3: Globotriaosylceramide; IS: Internal standard; nd: Not detected.
<b>Figure 5.</b>
Figure 5.. Plasma biomarker profile comparison for globotriaosylceramide isoforms/analogs of males (n = 9) and females (n = 9) nonobese diabetic/Severe Combined Immune Deficiency/Fabry mice and control mice (n = 5).
Table results are expressed as the area of each compound/area of the Gb3[(d18:1)(C18:0)D3] IS, which is the response factor for each biomarker. The number before each biomarker refers to: Group 1: Gb3 isoforms with saturated fatty acids; Group 2: Gb3 isoforms/analogs with one extra-double bond (on the sphingosine or fatty acid); Group 3: Gb3 isoforms/analogs with two extra-double bonds (on the sphingosine and fatty acid or both on the fatty acid); Group 4: Gb3 isoforms hydroxylated fatty acid; and Group 5: methylated Gb3 isoforms. *For Gb3 isoforms/analogs with one or two extra-double bonds, the isoforms with the extra-double bond(s) on the fatty acids were analyzed together with their structural isomers with one extra-double bond on the sphingosine moiety (analogs). T: Trace, which is <0.05. Error bars correspond to the mean plus one standard deviation. Gb3: Globotriaosylceramide; IS: Internal standard; nd: Not detected.
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