Analysis of sphingoid backbones 41 HPLC on sphingoid backbones

The measurement of sphingoid backbones is most prevalently done by derivat-ization and separation via high performance liquid chromatography (HPLC) (8). This method of analysis requires a mild alkaline hydrolysis of the lipids. The sphingoids are then derivatized on their free amine group (only available on the sphingoid backbones) with ori/zo-phthaldialdehyde (o-PA), a fluorescent compound. The samples are then injected and separated on HPLC, yielding a peak area for each sphingoid. The sensitivity of this system is around 5 pmol. This method allows for the separation of sphingoid backbones by their saturation, chain length, and, to a certain extent, stereochemistry {Figure 4a).

MINS

MINS

Figure 4. (a) HPLC chart of sphingolipid standards as follows: peak 1, l-threo sphingosine (11.6 min); peak 2, d-erythro sphingosine (12.3 min); peak 3, l-threo dihydrosphingosine (15.2 min); peak 4, d-erythro dihydrosphingosine (16.9 min); peak 5, C20 sphingosine (29.5 min), (b) HPLC chart of sphingoid backbones extracted from MOLT4 cells. Peak 1 is sphingosine (12.3 min), peak 2 is d -erythro dihydrosphingosine (16.5 min), and peak 3 is the internal standard C20 sphingosine (29.2 min). The front peak on each chart is un-reacted o-PA. Times given are the retention times, which can change with each run due to variation in columns, different back pressures, and slight differences in the running buffer.

Stereoisomers can be separated on a reverse phase column, but diastereomers require a chiral column. A further way of comparison available for this analysis is the use of an internal standard. An internal standard should ideally be a non-endogenous sphingoid backbone. Addition of the internal standard allows for comparison of samples within each experiment. 200 pmol of internal standard need to be added to every sample by step 5 of either the Folch (Protocol 2) or Bligh and Dyer (Protocol 1) extraction given above. In addition to the internal standard, the running of samples of specific sphingoid backbones is wise owing to possible shifts in retention times. Retention times shift because of the different reverse phase C18 columns used, back pressure changes, minor variances in the solvent systems, and type of HPLC system. Knowing the exact retention time of known standards allows for the selection of the proper peaks from mammalian samples (Figure 4b) from the background peaks.

Protocol 5. HPLC analysis of sphingoid backbones

Reagents

• D-erythro sphingosine and D,L-erythro dihydrosphingosine (Sigma)

. 3% K-borate buffer (pH 10.5 by addition of KOH pellets to boric acid solution)

Method

1. Prepare 100-200 pmol of known standards of d-erythro sphingosine and d,l-erythro dihydrosphingosine for derivatization. These standards are useful for defining retention times within an HPLC run. If necessary a standard curve of values can be constructed with known standards of the d -erythro sphingosine.

2. On the day of running the samples on HPLC, one should make up the o-PA reagent fresh. This requires 9.9 ml of 3 % K-borate buffer, 5 n,l of p-mercaptoethanol and 0.1 ml of 5% o-PA in 100% ethanol (for example, weigh out over 5 mg of o-PA and resuspend in 20 times the volume of weighed material in jjlI, i.e. 8.6 mg would be resuspended in 172 |xl of ethanol).

3. In order to derivatize dried down lipids from the alkaline hydrolysis procedure (Protocol 3) they must be resuspended in 50 jjU of methanol and then reacted with 50 n,l of the freshly made o-PA solution. The samples should be incubated for at least 5 min in the dark as the reagent is light sensitive (note: samples should be kept in the dark throughout the HPLC run).

4. Add 0.2-0.5 ml of the running buffer to each tube.

• fS-mercaptoethanol

• running buffer: 90% methanol, 10% 5 mM potassium phosphate, pH 7.0

5. Detection on HPLC is at an emission wavelength of 455 nm with excitation wavelength of 345 nm using a fluorescence detector.

6. Samples can now be injected on a reverse phase C18 column with a flow of 1 ml/min.

The quantitation of sphingoid backbones can either be done as a ratio to the internal standard and then normalized to phosphate, or in absolute amounts with the suggested external standard curve then normalized to phosphate. The case of using the ratio of experimental lipid over internal standard is done by simply dividing the peak area of experimental lipid by the peak area of the known standard. This gives a number that takes into consideration the extraction efficiency throughout the procedure and, once normalized to lipid phosphate, can be easily compared between control and treated samples. The reason for this is that the ratio does not change throughout experimental manipulations such as injections and quantitative transfers. Use of the external standard curve involves more manipulation. First, the peak area must be converted into picomoles via the standard curve generated. Next, one should determine the factors of how much sample was finally injected on the HPLC of the original third used. Therefore, one must account for extraction efficiency using the internal standard, amount injected over total amount, and any loss of sample through the quantitative transfer from the alkaline hydrolysis procedure. Once all these factors are accounted for one needs to calculate the true picomoles of each sphingoid backbone in each sample third and then normalize it to lipid phosphate. The absolute numbers can now be compared between control and treated samples.

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