Simultaneous Quantification of Cardiolipin, Bis(monoacylglycero)phosphate and their Precursors by Hydrophilic Interaction LC−MS/MS Including Correction of Isotopic Overlap

Cardiolipin (CL) and bis(monoacylglycero)phosphate (BMP) are unique lipid structures with important biological roles for mitochondrial integrity and endolysosomal degradation, respectively. They are synthesized from common precursors, phosphatidylglycerol (PG) and phosphatidic acid (PA). Here we present a rapid method for the simultaneous quantification of BMP, CL, PG, and PA using hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC−MS/MS). HILIC provides coelution of lipid species and their internal standards required for accurate quantification. Coelution leads to isotope overlap of lipid species which was successfully corrected. This assay was validated in mouse heart tissue and primary human skin fibroblasts. It shows reproducibility and limits of detection sufficient for biomarker studies contributing to basic research on BMP and CL metabolism

Characteristics of the study groups.

<p>BMI: Body Mass Index, LDL: low density lipoprotein, HDL: high density lipoprotein, VLDL: very low density lipoprotein.</p><p>*vs. control</p><p>* <i>p<</i>0.05</p><p>** <i>p<</i>0.001</p><p>*** <i>p<</i>0.0001</p><p>≠ vs. risk: ≠ <i>p<</i>0.05</p><p>≠ ≠ ≠ <i>p<</i>0.0001.</p><p>As expected, BMI, plasma glucose, triglycerides, HDL-cholesterol, VLDL-cholesterol and sCD163 are significantly different between healthy controls and MetS patients.</p

Correlation between BMI, sCD163 and triglycerides with total and individual SPC species.

<p>*. Correlation is significant at the 0.05 level (2-tailed), and</p><p>**. Correlation is significant at the 0.01 level (2-tailed).</p><p>Correlation between BMI, sCD163 and triglycerides with total and individual SPC species.</p

Correlation between BMI and sCD163 with S1P.

<p>*. Correlation is significant at the 0.05 level (2-tailed).</p><p>Correlation between BMI and sCD163 with S1P.</p

Individual LPC and PC P species after lipid loading.

(A) Individual LPC species on day five show saturation and lipoprotein dependent concentration changes (B) Modulation of PC P species after lipoprotein loading and subsequent 24h of HDL3 mediated deloading on experimental day six. For individual PC P only species with detectable levels are shown. n = 9 for all experiments except n = 8 for 6d MCSF control data., means +/- SD. (* p<0.05, ** p<0.01, *** p<0.001).</p

Flow cytometric analysis of M1/M2 surface markers in lipoprotein-treated macrophages.

CD163 (scavenger receptor cysteine-rich type 1 protein M130; hemoglobin/haptoglobin receptor) and CD206 (C-type 1 lectin receptor; mannose receptor) were used as M2 markers while CCR7 (CD197) and CD11c served as M1 markers. Loading with eLDL only induced a statistically significant decrease in CD11c surface expression. On the other hand loading with oxLDL led to significant decreases in CD163 and CD11c, as well as to increases in CCR7 and a not statistically significant decrease in CD206. n = 6, means +/- SD; (*p<0.05, ** p<0.01, *** p<0.001).</p

Estimation of membrane fluidity by calculation of lipid ratios on day six.

(A) The ratios of saturated to mono-unsaturated PE P species and (B) of saturated to poly-unsaturated PE Ps were calculated as markers for membrane fluidity in control and treated cells on day six. Additionally the mean PC to FC ratio was determined (C). Means +/- SD, n = 9 for control and deload measurements and n = 8 for loaded cells; (a: p<0.001 against control and eLDL loaded cells, b: p<0.001 against control cells, c: p<0.05 against controls, d: p<0.01 against controls).</p

Plasma levels of SPC and S1P from controls and patient groups.

<p>(A): Total SPC; (B): SPC-species; (C): S1P. SPC levels were significantly increased in MS patients but not in those at risk for diabesity (A). SPC increase was due to an increase in all three main subspecies (B). MetS patients have significant lower levels of S1P (C). S1P and SPC-species (n = 8 (Control), n = 8 (Risk), and n = 9 (MetS) were analyzed by LC-MS/MS and are expressed in μmol/l of plasma. Data presented as mean ± SEM. * <i>p</i><0.05, **<i>p</i><0.005, ***<i>p</i><0.0005 statistically different with respect to control.</p

Plasma levels of CE, PC, LPC, SM (A), and PE, PE P, PI, and Cer (B) from controls and patient groups.

<p>From all the major lipid classes analyzed only changes in LPC levels reached significance in MetS patients ((n = 12 (Control), n = 19 (Risk), and n = 33 (MetS)) (A). Lipid species were analyzed either by LC-MS/MS or ESI-MS/MS, as described in Methods, and are expressed in μmol/l of plasma. Data presented as mean ± SEM. * <i>p</i><0.05, statistically different with respect to control.</p

Hypothetical scheme depicting SPC and S1P synthesis.

<p>Ceramides (Cer) are generated from the sphingomyelinase pathway and other pathways such as the salvage pathway and the de novo synthesis pathway (not shown in this scheme). Sphingosine (SPH) which can be formed from the degradation and recycling of complex sphingolipids and glycosphingolipids in an acidic environment (salvage pathway) may also contribute to Cer metabolism. SPH is phosphorylated by SPH kinase to sphingosine-1-phosphate (S1P), a bioactive lipid intermediates with several effects. In addition to Cer, SPC is another biologically active lipid metabolite generated from sphingomyelin (SM) under the action of a SM-deacylase. Secreted SPC is likely a substrate for autotaxin (ATX), an exoenzyme with lysophospholipase D activity, which leads to S1P generation. Alternatively, S1P can be converted from SPC by ectonucleotide pyrophosphatase/phosphodiesterases (ENPPs). SPC and S1P can both induce their effects through binding to G-protein coupled receptors present on different cell types. It cannot be excluded that an intracellular SPC—> S1P conversion also occurs by a yet unidentified SPC-ase, and not SPC but rather S1P is secreted from cells predominantly. Extracellular S1P might be converted back to SPC through not yet identified enzyme(s). Substantial amounts of extracellular SPC and S1P are loaded to preβ-HDL particles and therefore they may contribute to the composition and/or maturation of α-HDL lipoproteins. UC: unesterified cholesterol, PC: phosphatidylcholine, LPC: lysophosphatidylcholine, PM: plasma membrane.</p