Quantitative determination of sphingomyelin II

Authors：

Category

Glycolipids and related compounds

Protocol Name

Authors

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

Ito, Makoto
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University

*To whom correspondence should be addressed.

KeyWords

Sphingomyelin Alkaline phosphatase Choline oxidase Sphingomyelinase Ceramide

Reagents

●	Reaction buffer: 50 mM Tris-HCl, pH 7.4, containing 5 mM MgCl₂
●	Standard dilution buffer: 50 mM Tris-HCl, pH 7.4, containing 2% Triton X-100 and 5 mM MgCl₂
●	12.5 U/mL of Bacillus cereus sphingomyelinase (Sigma-Aldrich, St. Louis, MO, #9396)
●	400 U/mL of calf intestinal alkaline phosphatase (GE Healthcare, Little Chalfont, UK, E2250Y)
●	120 U/mL of choline oxidase (Sigma-Aldrich, #26986)
●	200 U/mL of horseradish peroxidase (Sigma-Aldrich, #77334)
●	20 mM of Amplex Red (Invitrogen/Life Technologies, Carlsbad, CA, #A12222) [Note] All stock solutions are prepared in the reaction buffer except Amplex Red, which is in DMSO.
●	C18-sphingomyelin (Matreya LLC, Pleasant Gap, PA, #1911): Make a 20 mM C18-sphingomyelin stock solution in ethanol and keep at −20°C.
●	Enzyme cocktail: 12.5 mU of Bacillus cereus sphingomyelinase, 400 mU of alkaline phosphatase, 120 mU of choline oxidase, 200 mU of horseradish peroxidase, and 20 nmol of Amplex Red reagent in 100 μL of reaction buffer

Instruments

●	96-well micro plate (Black plate)
●	ARVO fluorescence microplate reader (PerkinElmer, Waltham, MA)

Methods

Quantitative determination of sphingomyelin [II]

1)	Homogenize the sample in a 20-fold volume of 0.2% Triton X-100.	Comment 0

2)	Centrifuge at 10,000 × g for 5 min.	Comment 0

3)	Withdraw the supernatant.	Comment 0

4)	Add 100 μL of enzyme cocktail to each well of a 96-well microtiter plate.	Comment 0

5)	Add 5 μL of sample to each well.	Comment 0

6)	For controls, add 5 μL of the sample to the enzyme cocktail without sphingomyelinase.	Comment 0

7)	Incubate at 37°C for 20 min.	Comment 0

Measure the fluorescence of the sample in the microtiter plate with a microplate reader (Set excitation and emission wavelengths at 544 and 590 nm, respectively).

Comment 0

Notes

Sphingomyelin content is calculated from the test value after subtracting the control value.

Standard sphingomyelin: 2, 4, 10, 20, 40, 100, 200, 400 pmol/μL (10, 20, 50, 100, 200, 500, 1000, 2000 pmol/5 μL/well)

Discussion

With this procedure, 20–1000 pmol/well of sphingomyelin can be determined.

Figure & Legends

Fig. 1. Scheme for sphingomyelin determination through four enzymatic steps

(A) Quantification of ceramide using diacylglycerol kinase

(B) Quantification of ceramide using ceramide kinase

(D) Quantification of sphingomyelin through four enzymatic steps

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Date of registration：2014-07-31 10:42:46