Novel Procedure for Measuring Psychosine Derivatives by an HPLC Method

Abstract

We developed a sensitive and simple method to determine galactosylsphingosine and glucosylsphingosine as a 4-fluoro-7-nitrobenzofurazan autofluorescent compound, using HPLC equipped with a Showdex sugar column. Amounts of galactosylsphingosine were successfully measured in the picomole range. This novel procedure is more stable and simpler than the previous method using o-phthalaldehyde. It was applied to tissues from the twitcher mouse, an animal model of human globoid cell leukodystrophy. The amount of galactosylsphingosine was 34-102 micrograms/kg of wet tissues in control cerebrum and cerebellum, whereas in twitcher mice the range was 2,251-4,228 micrograms/kg of wet tissues. The psychosine concentration was also increased in the liver and kidney of twitcher mice, respectively, 1,513 micrograms and 1,106 micrograms/kg of wet tissue (normal liver, 125 micrograms; normal kidney, 74 micrograms/kg of wet tissue). This novel procedure is useful for the pathochemical evaluation of lysosphingolipids in various sphingolipidoses as well as in other neuropathological and cellular conditions.

Psychosine-induced alterations in peroxisomes of twitcher mouse liver

“…These results are consistent with previous published analysis [3,35,36], and support the notion that globoid cell leukodystrophy is a generalized psychosine storage disease.…”

“…The psychosine analysis in various tissues of Krabbe patients and twi mouse indicate that levels in nervous tissue are much higher than in other organs [3]. However, as psychosine also accumulates in peripheral organs (liver, kidney and lungs amongst others) [3,35,36], globoid cell leukodystrophy has been referred to as a generalized psychosine storage disease [3].…”

Direct quantitation of psychosine from alkaline-treated lipid extracts with a semi-synthetic internal standard

“…Figure 6 shows a series of representative ESI MS/MSs of psychosine in lipid extracts from spinal cord, cortex, cerebellum, and brain stem acquired by using NL scanning of 180 amu of 1-month-old mice. The results from mice at 1 month of age are in good agreement with those from other studies using different methods (12,14), although determination of the psychosine content in brain samples of 5.5-month-old mice was not found in the literature. Intriguingly, the distribution of psychosine closely paralleled the expression levels of sphingosine-1-phosphate receptors (30)(31)(32)(33), indicating a correlation of psychosine with its receptor distribution.…”

“…Previous methods were largely based on chromatography, either TLC or HPLC, with or without chemical modifications (8)(9)(10)(11)(12)(13)(14). The drawbacks of these methods include the usage of radioactive reagents, the labor intensiveness of the procedures, relatively low sensitivity of detection, or the lack of the structural information.…”

Glucosylsphingosine Accumulation in Mice and Patients with Type 2 Gaucher Disease Begins Early in Gestation

“…The quantitation of Glc-sph was achieved by HPLC separation of NBD-F-generated autofluorescent derivatives (22) that were detectable in the picomole range. Linearity was demonstrated over concentrations ranging 100-fold.…”