Analysis of sphingomyelins

Presented in this section are two methods for the analysis of sphingomyelin. The first method is one of radiolabelling the sphingomyelin pool with tritiated choline, extracting the lipids, performing alkaline hydrolysis, and then measuring total sphingomyelin by counts released by incubation with bacterial sphingomyelinase (11) from Streptomyces (Sigma). The advantage of this method is the number of samples that may be analysed quickly; however, the method does not allow for analysis of particular sphingomyelins. The second method follows the same initial work-up as the first but then separates the sphingomyelins via HPLC (12) and fractions are collected and then counted. The advantage of this method is specific types of sphingomyelins (Figure 6) can be followed, but each sample takes a long time to run and count.

To quantitate the results one must calculate total cpms in each tube. Since we took 400 p,l of the 800 jjuI upper phase (the upper phase is everything except chloroform), multiply the cpms by two. This value can now be

Figure 6. HPLC profiles of brain sphingomyelins. Peak 1 is 16:0 fatty acyl SM (24 min), peak 2 is 18:0 fatty acyl SM (31 min), peak 3 is 20:0 fatty acyl SM (47 min), peak 4 is a combination of 22:0 fatty acyl SM and 24:1 unsaturated fatty acyl SM (68 min), and peak 5 is 24:0 fatty acyl SM (85 min).

Mins

Figure 6. HPLC profiles of brain sphingomyelins. Peak 1 is 16:0 fatty acyl SM (24 min), peak 2 is 18:0 fatty acyl SM (31 min), peak 3 is 20:0 fatty acyl SM (47 min), peak 4 is a combination of 22:0 fatty acyl SM and 24:1 unsaturated fatty acyl SM (68 min), and peak 5 is 24:0 fatty acyl SM (85 min).

normalized to the phosphate value obtained above. Total sphingomyelin levels often decrease up to 30% with certain treatments.

Protocol 7. Bacterial sphingomyelinase on total SM

Reagents

• TMT buffer: 0.19 M Tris-HCI, pH 7.5, 12 mM .bacterial SMase: 2 units/ml in 10 mM MgCI2, and 0.2% Triton X-100 Tris-HCI, pH 7.5

Method

1. Suspend samples to be tested in 50 n-l of TMT buffer and vortex vigorously.

2. Sonicate the samples for three one-minute bursts, vortex after each sonication then rest for 1 min between each sonication.

3. Pre-incubate the samples at 37°C for 5 min.

4. Add 50 (xl of bacterial Smase to each tube and allow the reaction to proceed for 30 min at 37 °C.

5. Stop the reaction with 1.5 ml of chloroform/methanol and vortex.

6. Add 0.2 ml of water to induce a phase break in the sample and vortex.

7. Spin tubes at 2000 g for 5 min to clarify phases.

8. Collect 400 (jil of the upper phase for scintillation counting. (Remember, the cells were labelled with precursors to the polar head group and once incorporated will be released to the aqueous phase by enzyme cleavage.)

Protocol 8. H PLC on SM

Method

1. Prepare a known standard of radiolabeled SM for a control run.

2. Take samples post-extraction and alkaline hydrolysis and resuspend them in methanol.

3. Inject samples on to a C18 reverse phase column at a flow rate of 1 ml/min in a running system of 98% methanol, 2% 5 mM potassium phosphate, pH 7.0.

4. Count by either fraction collecting and scintillation counting or a flow through detector.

After counting each fraction a chart can be prepared for each sample. The charts of a treated versus non-treated sample can now be directly compared to see which specific SM species is changing. This comparison will allow analysis of each peak for changes in a specific sphingomyelin type.

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