Quantitative determination of sphingomyelin

Quantitative determination of sphingomyelin [I]

a.

n-Octylglucoside (#494459, Merck Millipore, Billerica, MA)

b.

Cardiolipin (C0563, Sigma-Aldrich, St. Louis, MO)

c.

Diethylenetriaminepentaacetic acid (DETAPAC) (D1133, Sigma-Aldrich)

d.

Sphingomyelin standard (C18-sphingomyelin, #1911, Matreya LLC, Pleasant Gap, PA): Make a 20-mM C18-sphingomyelin stock solution in ethanol and keep at −20°C.

e.

Bacillus cereus sphingomyelinase (S9396, Sigma-Aldrich)

f.

E. coli DGK (D3065, Sigma-Aldrich)

g.

Chloroform (CHCl₃)

h.

Methanol (MeOH)

i.

CHCl₃-MeOH mixture (C/M, volume/volume)

j.

0.1 M Imidazol-HCl, pH 6.6

k.

Distilled water (DW)

l.

Thin-layer chromatography (TLC) plates (Silicagel 60, 20 × 20 cm, Merck Millipore)

Quantitative determination of sphingomyelin [II]

a.

Standard sphingomyelin (C18-sphingomyelin, #1911, Matreya LLC): Make a 20-mM C18-sphingomyelin stock solution in ethanol and keep at −20°C.

b.

Bacillus cereus sphingomyelinase (S9396, Sigma-Aldrich)

c.

Calf intestinal alkaline phosphatase (2250A, Takara Bio Inc., Otsu, Japan)

d.

Choline oxidase (#26986, Sigma-Aldrich)

e.

Horseradish peroxidase (HRP, #77334, Sigma-Aldrich)

f.

Amplex Red Reagent (A12222, Invitrogen/Life Technologies, Carlsbad, CA): Make a 20-mM stock solution in dimethyl sulfoxide (DMSO) and keep at −20°C (Note 1)

g.

Enzyme cocktail: 12.5 mU of B. 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 the reaction buffer

h.

Reaction buffer: 50 mM of Tris-HCl, pH 7.4, which contains 5 mM of MgCl₂

i.

Standard dilution buffer: 50 mM of Tris-HCl, pH 7.4, which contains 2% Triton X-100 and 5 mM of MgCl₂

j.

96-Well microplate (Black plate)

Imaging analyzer (FLA 5000, Fujifilm, Tokyo, Japan)

Sonic bath

Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA)

ARVO fluorescence microplate reader (PerkinElmer, Waltham, MA)

Quantitative determination of sphingomyelin [I]

a.

Dissolve lipids whose sphingomyelin content is measured in 10 μL of 7.5% n-octyl-β-D-glucopyranoside, 5 mM of cardiolipin, and 1 mM of DETAPAC.

b.

Mix with 30 μL of 0.1 M imidazole–HCl, pH 6.6, containing 0.1 M of NaCl, 25 mM of MgCl₂, 4 mM of DTT, and 2 mM of EGTA.

c.

Add 5 μL of enzyme (5 mU of E. coli DGK and 100 mU of B. cereus sphingomyelinase) in 10 mM of imidazole–HCl, pH 6.6, containing 1 mM of DETAPAC, 1 mM of DTT, and 10% glycerol.

d.

Initiate the reaction by adding 5 μL of 5 mM “cold” adenosine triphosphate (ATP) and 0.1 μCi of [γ-³²P]ATP in the buffer described in Step c (the final 50-μL reaction mixture contains 0.5 mM of ATP and 0.1 μCi of [γ-³²P]ATP).

e.

Incubate at 25°C for 1 h.

f.

Add 0.6 mL of CHCl₃/MeOH (1/1, v/v).

g.

Vortex for a few seconds, and add 250 μL of 1 M KCl in 20 mM of Mops, pH 7.0.

h.

Centrifuge at 10,000 ×g for 5 min.

i.

Transfer the organic phase to an Eppendorf tube and dry under a stream of N₂ gas.

j.

Dissolve the sample in 20 μL of CHCl₃/MeOH (1/1, v/v), and apply an aliquot of the solution on a TLC plate.

k.

Develop the TLC plate with CHCl₃/acetone/ MeOH /acetic acid/water (10/4/3/2/1, v/v/v/v/v).

l.

Dry the TLC plate, and cover it with a wrap.

m.

Expose the TLC plate to an imaging plate.

n.

Determine the radioactivity of the bands corresponding to [³²P]Cer-1-P with an imaging analyzer, such as the FLA5000 (Note 2).

Quantitative determination of sphingomyelin [II]

a.

Homogenize a sample whose sphingomyelin content is measured in a 20-fold volume of 0.2% Triton X-100.

b.

Centrifuge at 10,000 ×g for 5 min.

c.

Withdraw the supernatant.

d.

Add 100 μL of enzyme cocktail to each well of a 96-well microtiter plate.

e.

Add 5 μL of the sample homogenate to each well.

f.

For controls, add 5 μL of the sample homogenate to the enzyme cocktail without sphingomyelinase.

g.

Incubate at 37°C for 20 min.

h.

Measure the fluorescence in the wells of microtiter plate with a microplate reader (set excitation and emission wavelengths at 544 and 590 nm, respectively) (Notes 3–5).

All stock solutions are prepared in the reaction buffer except Amplex Red, which is in DMSO.

To determine the sphingomyelin content of the samples, the amount of endogenous ceramide, determined in a control experiment wherein the reaction is conducted without Bacillus sphingomyelinase, should be subtracted. The quantification of sphingomyelin can be determined from the standard curve using a known amount of standard sphingomyelin (C18-sphingomyelin). With this procedure, 10–1,000 pmol of sphingomyelin can be determined.

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

Quantification of sphingomyelin can be determined from the standard curve using a known amount of standard sphingomyelin (C18-sphingomyelin): 2, 4, 10, 20, 40, 100, 200, and 400 pmol/μL (10, 20, 50, 100, 200, 500, 1,000, and 2,000 pmol/5 μL/well).

Using this procedure, 20–1,000 pmol/well of sphingomyelin can be determined.