Quantification of ceramides

Quantification of total ceramides

After separation by column chromatography or better by TLC, ceramides are derivatized with a UV absorbing or a fluorescent tag before HPLC.

A- Low sensitivity measurements:

When sufficient amounts of ceramides are available (more than about 5 or 10 µg), it is convenient to use a UV absorbing tag to quantify these lipids.

Reagents:

dry pyridine, methanol, hexane, 2-propanol
Methanol saturated with Na₂CO₃, 0.6M HCl
Benzoyl chloride

Procedure:

Derivatization:

Ceramides are dried and dissolved in 20 µl pyridin then 40 µl benzoyl chloride are added. Heat 1 h at 60°C.
Evaporate the solution under an efficient fume hood and add 4 ml methanol. Heat again 1 h at 70°C. Evaporate the methanol phase, add 1 ml hexane and 1 ml Na₂CO₃ saturated methanol solution. Vortex and centrifuge at low speed.
The lower phase is washed first with 1 ml methanol containing 0.6 HCl and then with pure methanol. The lower phase is evaporated and the residue is dissolved iin a known volume of hexane before analysis by HPLC.

Quantification by HPLC:

Column: Lichrosorb Si 60 from Merck (125 x 4 mm, 5 µm)
Mobile phase: hexane/2-propanol (98/2, v/v), flow rate: 0.5 ml
Detection: UV at 240 nm
Retention time: about 5 min

B- High sensitivity measurements:

When the amount of ceramides is lower than 5 µg it is recommended to use a fluorescent tag as naproxen which enables also an accurate determination of molecular species composition.

Derivatization of ceramides:

The procedure is the same as that used for diacylglycerols.

Quantification by HPLC:

Column: Lichrosorb Si 60 from Merck (125 x 4 mm, 5 µm)
Mobile phase: acetonitril/2-propanol (70/30, v/v), flow rate: 1 ml
Fluroscence detection: excitation: 230 nm, emission: 352 nm.
Retention time: about 2 min.

Other methods:

As a highly specific recombinant ceramide kinase was cloned, characterized and prepared, a sensitive, rapid and specific enzymatic method for cellular lipid extracts may be adopted (Bektas M et al., Anal Biochem 2003, 320, 259). The new ceramide kinase assay is more specific than the diacylglycerol kinase method and eliminates the need for analysis of the lipid product by TLC.