The comparison of control versus treated samples is of utmost importance in the analysis of in vivo sphingolipids. Therefore, a method to ensure consistency between samples is necessary. The most commonly used is that of lipid phosphate measurement taken directly from the aforementioned sphingolipid extraction methods. Furthermore, lipid phosphate can be used to more accurately quantitate sphingolipid samples. This method normalizes for any variance in cell number and extraction efficiency, since lipid phosphate should not vary much between cells. The method relies upon a standard curve analysis and uses a colorimetric assay of phosphate that has been ashed.
Protocol 4. Measurement of lipid phosphate
. ashing buffer: H20:10 N H2S04:70% HCI04 • 10% w/v L-ascorbic acid (40:9:1)
1. Prepare a standard curve of phosphates in duplicate. Put 0, 3, 5, 7, 10, 12, 15, 20, and 30 )xl of NaH2P04 (1 nmol. per |xl) in labelled tubes.
2. Dry down by speed vacuum or dry nitrogen the aliquots from either extraction above. Use the same style of glass tube for both the standards and the samples to ensure consistent ashing.
3. Add 0.6 ml of ashing buffer and vortex.
4. Place samples In a heating block at 160°C overnight. Approximately 100 |xl should be left in the tube after incubation.
5. Add 0.9 ml of water and mix after allowing the samples to cool.
6. Now add 0.5 ml of ammonium molybdate and add 0.2 ml of L-ascorblc acid. Mix tubes very vigorously.
8. Read samples on a spectrophotometer at 820 nm in 0.8 ml cuvettes. First construct the standard curve then use it to get the experimental values.
The number obtained from the phosphate measurement now becomes the denominator for the samples. This means that results are expressed as pico-moles of sphingolipids/nanomoles of lipid phosphate. This will be accomplished by comparing the value of the third obtained above to the value of the measurement obtained in the experimental third of sphingolipids. However, phosphate levels should be used to normalize sphingolipids for comparison of cells from a given cell line and not for comparison between cell lines. Cell lines can have very different lipid phosphate levels per cell. Therefore, analysis should be limited to time-matched controls with a change in only one variable.
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