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Simultaneous quantification of glucosylceramide and galactosylceramide by HPLC

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Category
Glycolipids and related compounds
Protocol Name

Simultaneous quantification of glucosylceramide and galactosylceramide by HPLC

Authors
 Kota Zama *
Biofunctional Lipid Biology, Frontier Research Center for Post-genome Science and Technology, Hokkaido University

Nozomu Okino
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University

Makoto Ito
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
Keywords

glucosylceramide

galactosylceramide

sphingolipid ceramide N-deacylase

high-performance liquid chromatography

Reagents
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GlcSph (bovine) (Matreya)

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GalSph (bovine brain) (Alexis Biochemical)

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NBD-GlcCer (Sigma)

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Psudomonas SCDase (Takara Bio.)

Instruments
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Normal-phase column (Intersil SIL 150A-5, 4.6 X 250 mm) (GL science)
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Fluorescent detector (Hitachi L-7840)
Methods
1.

Extraction of GlcCer and GalCer

1) 

 Add 400 μl of chloroform/methanol (1/1, v/v) and 20 μl of 1 μM C6-NBD-GlcCer (internal standard) to cell lysate (30 μl), and keep at 37°C for 2 h.

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2) 

 Add 200 μl of chloroform and 150 μl of water and mix well.

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3) 

 Withdraw the lower phase (chloroform layer) after centrifugation at 15,000 rpm for 5 min.

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4) 

 Add 200 μl of methanol and 150 μl of water to the lower phase.

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5) 

 Withdraw the lower phase after centrifugation and dry using a speed Vac concentrator. The lower phase (chloroform phase) is recovered as the GSL fraction which contains GlcCer and GalCer.

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2.

SCDase treatment and OPA derivatization

1) 

 Add SCDase (0.6 mU in 3 μl) to the sample dissolved in 27 μl of 25 mM sodium acetate buffer, pH 5.5, containing 5 mM CaCl2 and 2.0% TritonX-100.

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2) 

 Incubate at 37oC for an appropriate period. Stop the reaction with 200 μl of chloroform/methanol (1:1, v/v).

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3) 

 Add water (15 μl) to the chloroform/methanol solution, mix well, and centrifuge.

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4) 

 Withdraw the lower phase (chloroform phase).

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5) 

 Add chloroform (200 μl) to the upper phase, centrifuge, and withdraw the lower phase. Repeat steps 3)-5) twice.

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6) 

 Pool the lower phases from the 3 extractions, dry with a Speed Vac concentrator, and dissolve in 120 μl of ethanol.

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7) 

 Mix the ethanol solution, and OPA reagent [0.1 ml of ethanol containing 10 mg of OPA, 20 μl of 2-mercaptoethanol, and 9.9 ml of 3 % (w/v) boric acid buffer, pH 10.5] , and incubate at 70oC for 20 min.

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8) 

 Add OPA reagent (15 μl) to the ethanol solution and keep at 70oC for 60 min.

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9) 

 Centrifuge the sample at 15,000 rpm for 10 min and transfer the supernatant to a glass vial.

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10) 

 Inject an aliquot of sample (15 μl) into an HPLC column using an auto-sampler.

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3.

HPLC analysis

1) 

 Analyze the OPA-derivatized sample with C6-NBD-GlcCer (internal standard) on a normal-phase column (intersil SIL 150A-5, 4.6 x 250 mm, GL science, Japan) using n-hexane/isopropylalchol/H2O (73/26.5/0.5, v/v/v) as a mobile phase at a flow rate of 2.0 ml/min and detect the OPA-derivatives using a fluorescent detector (Hitachi L-7840) set to excitation and emission wavelengths of 340 nm and 455 nm. OPA-GlcSph and OPA-GalSph are eluted at 6.8 min and 11.3 min, respectively.

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2) 

 Under the conditions described in 1), the internal standard is not detected. To quantify the internal standard, inject an aliquot of sample into the column and elute with n-hexane/isopropylalchol/H20 (44:55:1, v/v/v) at the flow rate described in 1). C6-NBD-GlcCer is eluted at 3.5 min as detected by a fluorescent detector set to excitation and emission wavelengths of 470 nm and 530 nm.

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Notes
  1. The hydrolysis of both GlcCer and GalCer by SCDase proceeds quantitatively from 5 pmol to 10 nmol under the conditions described above.
  2. Linearity for the determination of GlcCer and GalCer is observed from 5.0 - 450 pmol and 4.0 - 800 pmol in biological samples, respectively, which corresponds to approximately 103 to 105 RPMI cells and 5 to 80 zebrafish embryos.
  3. Prepare chloroform/methanol solutions just before use.
  4. Be careful to partition the sample with chloroform/methanol. Do not mix the lower phase with the upper phase, as this may disturb the baseline of HPLC.
  5. To remove contaminants such as Triton X-100 from the HPLC column, n-hexane/isopropylalchol/H20/phosphoric acid (100:60:5.7:0.3) is recommended.
  6. To hydrolyze GlcCer and GalCer by SCDase, extracted lipids should be completely dissolved in reaction buffer by sonication.
Figure & Legends

Figure & Legends

Fig. 1. Outline of the quantification of GlcCer and GalCer.

This figure was originally published in Glycobiology. 19(7):767-75. 2009 "Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase" Zama K, Okino N, Ito M. et al. Oxford University Press.

 

 

Fig. 2. Separation of OPA-GlcSph and OPA-GalSph by normal-phase HPLC.

This figure was originally published in Glycobiology. 19(7):767-75. 2009 "Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase" Zama K, Okino N, Ito M. et al. Oxford University Press.

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