Analysis of Fluorescent Ceramide and Sphingomyelin Analogs: A Novel Approach for in Vivo Monitoring of Sphingomyelin Synthase Activity

Abstract

A novel sensitive high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method was developed for real-time monitoring of relative sphingomyelin synthase (SMS) activity based on the measurement of a fluorescent ceramide (Cer) analog and its metabolite, a fluorescent sphingomyelin (CerPCho) analog, in plasma. Analyses were conducted using HPLC-FLD following a protein precipitation procedure. The chromatographic separations were carried out on an Agilent C18 RP column (150 × 4.6 mm, 5 μm) based on a methanol-0.1 % trifluoroacetic acid aqueous solution (88:12, by vol) elution at a flow-rate of 1 mL/min. The limit of quantification in plasma was 0.05 μM for both the fluorescent Cer analog and its metabolite. Significant differences in the fluorescent Cer analog and its metabolite concentration ratio at 5 min were found between vehicle control group and three D2 (a novel SMS inhibitor) dose groups (P < 0.05). Dose-dependent effects (D2 doses: 0, 2.5, 5, 10 mg/kg) were observed. Our method could be used to detect relative SMS activity in biochemical assays and to screen potential SMS inhibitors in vivo. D2 was found to be a potent SMS inhibitor in vivo, and may have a potential antiatherosclerotic effect, which is under further study. D609 was also selected as another model SMS inhibitor to validate our newly developed method.

Discovery of 4-Benzyloxybenzo[d]isoxazole-3-amine Derivatives as Highly Selective and Orally Efficacious Human Sphingomyelin Synthase 2 Inhibitors that Reduce Chronic Inflammation in db/db Mice

“…C6-NBD-ceramide (in 5% ethanol/5% Cremophor EL/0.9% NaCl aqueous solution) was injected into the tail vein at predose and at 1 and 2 h after administration. After 5 min, blood samples were drawn to measure SM synthase activity . SM synthase activity of ICR mice was measured according to the method described for measurement of SMS1 and SMS2 inhibitory activity in vitro .…”

“…After 5 min, blood samples were drawn to measure SM synthase activity. 43 SM synthase activity of ICR mice was measured according to the method described for measurement of SMS1 and SMS2 inhibitory activity in vitro. Test compounds (40 mg/kg) were intraperitoneal injected daily into a group of three male ICR mice (20−23g) for 7 days, and plasma samples were drawn to measure SM levels by the enzymatic measurement as reported previously.…”

“…After incubating at 37 °C for 30 min, the reaction was quenched with 200 μL of anhydrous ethanol. The mixture was then centrifuged at 7178 g for 10 min to produce the supernatant collected for the HPLC test 43. (b) The inhibitory activity of SMS2 was measured as follows: The assay was similar to the assay used for measuring SMS1 activity, except 79 μL of the SMS2 enzyme (0.0041 μg/μL) in DDM and 1 μL of a DMSO solution of the compound were used.…”

Sphingomyelin synthase 2 facilitates M2-like macrophage polarization and tumor progression in a mouse model of triple-negative breast cancer

“…The cells were centrifuged at 3000 × g for 10 min, and the lower phase was isolated, dried under a N 2 stream, and redissolved in 100 μL methanol. NBD-SM was detected by using HPLC as reported by Huang et al [37].…”