Determination of endogenous sphingolipid content in stroke rats and HT22 cells subjected to oxygen-glucose deprivation by LC‒MS/MS

Abstract

Background: Stroke is the leading cause of death in humans worldwide, and its incidence increases every year. It is well documented that lipids are closely related to stroke. Analyzing the changes in lipid content in the stroke model after absolute quantification and investigating whether changes in lipid content can predict stroke severity provides a basis for the combination of clinical stroke and quantitative lipid indicators.

Methods: This paper establishes a rapid, sensitive, and reliable LC‒MS/MS analytical method for the detection of endogenous sphingolipids in rat serum and brain tissue and HT22 cells and quantifies the changes in sphingolipid content in the serum and brain tissue of rats from the normal and pMCAO groups and in cells from the normal and OGD/R groups. Using sphingosine (d17:1) as the internal standard, a chloroform: methanol (9:1) mixed system was used for protein precipitation and lipid extraction, followed by analysis by reversed-phase liquid chromatography coupled to triple quadrupole mass spectrometry.

Results: Based on absolute quantitative analysis of lipids in multiple biological samples, our results show that compared with those in the normal group, the contents of sphinganine (d16:0), sphinganine (d18:0), and phytosphingosine were significantly increased in the model group, except sphingosine-1-phosphate, which was decreased in various biological samples. The levels of each sphingolipid component in serum fluctuate with time.

Conclusion: This isotope-free and derivatization-free LC‒MS/MS method can achieve absolute quantification of sphingolipids in biological samples, which may also help identify lipid biomarkers of cerebral ischemia.

Keywords: Content determination; Endogenous; Ischemic stroke; Liquid chromatography; Mass spectrometry; OGD/R-Induced; Sphingolipids.

Fig. 1

Verification of the pMCAO rat model and OGD/R HT22 model. a Determination of brain injury by TTC staining, b changes in weights of rats in normal and model groups, c cell viability detected by CCK-8, d inverted microscope to observe the morphological changes of HT22 cells after OGD/R injury

Fig. 2

Product ion mass spectra of sphingosine (d16:0) (A), sphinganine (d18:0) (B), phytosphingosine (C), sphingosine-1-phosphate (D) and sphingosine (d17:1) (IS, E)

Fig. 3

Chromatograms of sphingolipids and internal standard. A Methanol B 4% BSA C Standard solution (dissolved in methanol) D Brain tissue sample (without internal standard) E Serum tissue sample (without internal standard) F Cell sample (without internal standard) G Brain tissue sample (with internal standard) H. Serum sample (with internal standard) L. Cell sample (with internal standard). 1. Sphingosine-1-phosphate; 2. Phytosphingosine; 3. Sphinganine (d18:0); 4. Sphingosine (d17:1); 5. Sphinganine (d16:0)

Fig. 4

Changes in the contents of sphingosine-1-phosphate, phytosphingosine, sphinganine (d18:0), and sphinganine (d16:0) in the serum (A) and brain tissue (B) of SD rats in the normal and pMCAO groups and HT22 cells (C) in the normal and OGD/R groups (control, n = 6; model, n = 6). The data of each experiment are expressed as the mean ± standard deviation. Note. Compared with the normal or model group, *P < 0.05, **P < 0.01, ***P < 0.001