Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt(+/⁻) mice.
Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt(+/⁻) mice was quantified to be 406.06 ± 23.63 μg/g testis and 0.13 ± 0.02 μg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt⁻/⁻ males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt(+/⁻) males sire offspring. The higher than 50% expression level of SGG in Cgt(+/⁻) animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt(+/⁻) mice were sufficient for normal spermatogenesis.
Kongmanas
Kessiri
K
Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
Xu
Hongbin
H
Yaghoubian
Arman
A
Franchini
Laura
L
Panza
Luigi
L
Ronchetti
Fiamma
F
Faull
Kym
K
Tanphaichitr
Nongnuj
N
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D003124:Colorimetry / Q000379:methods; D005260:Female; D006017:Glycolipids / Q000032:analysis* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D051146:N-Acylsphingosine Galactosyltransferase / Q000378:metabolism; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013094:Spermatozoa / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013737:Testis / Q000378:metabolism
D006017:Glycolipids; C009233:seminolipid; D051146:N-Acylsphingosine Galactosyltransferase
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