Preparation of (13)C-labeled ceramide by acetic acid bacteria and its incorporation in mice

Abstract

We prepared 2-hydroxypalmitoyl-sphinganine (dihydroceramide) labeled with a stable isotope by culturing acetic acid bacteria with (13)C-labeled acetic acid. The GC/MS spectrum of the trimethylsilyl derivative of (13)C-labeled dihydroceramide gave molecular ions with an increased mass of 12-17 Da over that of nonlabeled dihydroceramide. The fragment ions derived from both sphinganine base and 2-hydroxypalmitate were confirmed to be labeled with the stable isotope in the spectrum. Therefore, (13)C-labeled dihydroceramide can be an extremely useful tool for analyzing sphingolipid metabolism. The purified [(13)C]dihydroceramide was administered orally to mice for 12 days, and the total sphingoid base fractions in various tissues were analyzed by GC/MS. The spectrum patterns specific to (13)C-labeled sphingoids were detected in the tissues tested. Sphinganine pools in skin epidermis, liver, skeletal muscle, and synapse membrane in brain were replaced by [(13)C]sphinganine at about 4.5, 4.0, 1.0, and 0.3%, respectively. Moreover, about 1.0% of the sphingosine pool in the liver was replaced by [(13)C]sphingosine, implying that exogenous dihydroceramide can be converted to sphingosine. These results clearly indicate that ingested dihydroceramide can be incorporated into various tissues, including brain, and metabolized to other sphingolipids.

Fig. 1.

Predicted metabolic pathway from [1-¹³C]acetic acid to [¹³C]dihydroceramide (2-hydroxypalmitoyl-sphinganine) in the biosynthesis of [¹³C]dihydroceramide by acetic acid bacteria. Acetic acid bacteria accumulate highly pure dihydroceramide as the main sphingolipid compound.

Fig. 2.

Analysis of the trimethylsilyl (TMS) derivatives of [¹³C]dihydroceramide and the sphingoid base by GC/MS. A: Mass spectrum of nonlabeled and ¹³C-labeled dihydroceramide analyzed by total ion monitoring (m/z 0–800). B: Relative intensities of nonlabeled and ¹³C-labeled dihydroceramide (m/z 750–780). C: Relative intensities of the sphingoid base (sphinganine) derived from hydrolyzed nonlabeled and ¹³C-labeled dihydroceramide (m/z 342–351) analyzed in selected ion monitoring mode.

Fig. 2.

Analysis of the trimethylsilyl (TMS) derivatives of [¹³C]dihydroceramide and the sphingoid base by GC/MS. A: Mass spectrum of nonlabeled and ¹³C-labeled dihydroceramide analyzed by total ion monitoring (m/z 0–800). B: Relative intensities of nonlabeled and ¹³C-labeled dihydroceramide (m/z 750–780). C: Relative intensities of the sphingoid base (sphinganine) derived from hydrolyzed nonlabeled and ¹³C-labeled dihydroceramide (m/z 342–351) analyzed in selected ion monitoring mode.

Fig. 2.

Analysis of the trimethylsilyl (TMS) derivatives of [¹³C]dihydroceramide and the sphingoid base by GC/MS. A: Mass spectrum of nonlabeled and ¹³C-labeled dihydroceramide analyzed by total ion monitoring (m/z 0–800). B: Relative intensities of nonlabeled and ¹³C-labeled dihydroceramide (m/z 750–780). C: Relative intensities of the sphingoid base (sphinganine) derived from hydrolyzed nonlabeled and ¹³C-labeled dihydroceramide (m/z 342–351) analyzed in selected ion monitoring mode.

Fig. 3.

Total ion chromatogram of the total sphingoid base fraction from murine muscle in GC/MS analysis. The total lipid of the tissue was hydrolyzed, applied for silica gel column chromatography. Eluate with chloroform:methanol (1:1, v/v) was collected and the trimethylsilyl (TMS) derivative of the total sphingoid base fraction was injected into GC/MS.

Fig. 4.

Total sphingoid base fractions of the tissues from [¹³C]dihydroceramide-administered mice. Trimethylsilyl (TMS) derivative of the sphingoid base was analyzed by GC/MS in selected ion monitoring mode. A: Relative intensities of sphinganine in epidermis, liver, skeletal muscle, and synapse membrane of the representative mouse (m/z 342–351). B: Relative intensities of sphingosine in the liver of the representative mouse (m/z 340–349).