Derivatization Strategy for the Comprehensive Characterization of Endogenous Fatty Aldehydes Using HPLC-Multiple Reaction Monitoring

Fatty aldehydes are crucial substances that mediate a wide range of vital physiological functions, particularly lipid peroxidation. Fatty aldehydes such as acrolein and 4-hydroxynonenal (4-HNE) are considered potential biomarkers of myocardial ischemia and dementia, but analytical techniques for fatty aldehydes are lacking. In the present study, a comprehensive characterization strategy with high sensitivity and facility for fatty aldehydes based on derivatization and high-performance liquid chromatography-multiple reaction monitoring (HPLC-MRM) was developed. The fatty aldehydes of a biosample were derivatized using 2,4-bis­(diethylamino)-6-hydrazino-1,3,5-triazine under mild and efficient reaction conditions at 37 °C for 15 min. The limit of detection (LOD) of the fatty aldehydes varied from 0.1 to 1 pg/mL, depending on the structures of these molecules. General MRM parameters were forged for the analysis of endogenous fatty aldehydes. “Heavy” derivatization reagents with 20 deuterium atoms were synthesized for both the discovery and comprehensive characterization of fatty aldehydes. More than 80 fatty aldehydes were detected in the biosamples. The new strategy was successfully implemented in global fatty aldehyde profiling of plasma and brain tissue of the bilateral common carotid artery (2VO) dementia rat model. Dozens of fatty aldehydes were significantly changed between the control and model groups. These findings further highlight the importance of endogenous fatty aldehydes

Correlation of sphingolipid biomarkers with ear swelling and splenic index.

<p>Correlation of sphingolipid biomarkers with ear swelling and splenic index.</p

Structure of triptolide.

<p>Structure of triptolide.</p

Visualization of sphingolipid metabolites in the plasma, kidney, spleen and liver.

<p>The figure depicts sphingolipid metabolites that participate in the early steps of sphingolipid biosynthesis. The pathway maps are overlaid by the fold changes in subclasses of sphingolipid metabolites between the control group and other groups by asterisks indicating the statistical significance on the right side (* = P<0.05, ** = P<0.01; n = 8). The content of each sphingolipid metabolite was determined as the mean of eight independent parallel samples.</p

Sphingolipid composition of mice kidney or plasma measured by triple quadruples MS/MS.

<p>Four groups including: control, model, model+triptolide and control+triptolide, each of which contains 8 samples. Sphingolipids were isolated from kidney homogenate corresponding to 1 mg protein or from 0.1 mL plasma. Bars are expressed as means ± SD, values for each sample are the average of 8 samples separately (pmol/mg protein, 0.1 mL plasma). Statistical difference from control or model group is indicated with an asterisk or a “&”, respectively. * or &: p<0.05 and ** or &&: p<0.01.</p

Sphingolipids as New Biomarkers for Assessment of Delayed-Type Hypersensitivity and Response to Triptolide

<div><h3>Background</h3><p>Hypersensitivity diseases are associated with many severe human illnesses, including leprosy and tuberculosis. Emerging evidence suggests that the pathogenesis and pathological mechanisms of treating these diseases may be attributable to sphingolipid metabolism.</p> <h3>Methods</h3><p>High performance liquid chromatography-tandem mass spectrometry was employed to target and measure 43 core sphingolipids in the plasma, kidneys, livers and spleens of BALB/c mice from four experimental groups: control, delayed-type hypersensitivity (DTH) model, DTH+triptolide, and control+triptolide. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify potential biomarkers associated with variance between groups. Relationships between the identified biomarkers and disease markers were evaluated by Spearman correlation.</p> <h3>Results</h3><p>As a treatment to hypersensitivity disease, triptolide significantly inhibit the ear swelling and recover the reduction of splenic index caused by DTH. The sphingolipidomic result revealed marked alterations in sphingolipid levels between groups that were associated with the effects of the disease and triptolide treatment. Based on this data, 23 potential biomarkers were identified by OPLS-DA, and seven of these biomarkers correlated markedly with the disease markers (p<0.05) by Spearman correlation.</p> <h3>Conclusions</h3><p>These data indicate that differences in sphingolipid levels in plasma and tissues are related to DTH and treatment with triptolide. Restoration of proper sphingolipid levels may attribute to the therapeutic effect of triptolide treatment. Furthermore, these findings demonstrate that targeted sphingolipidomic analysis followed by multivariate analysis presents a novel strategy for the identification of biomarkers in biological samples.</p> </div

Score plots from supervised OPLS-DA.

<p>Analysis showed distinct clustering between each group in the spleen, kidney, liver and plasma. R2Y(cum) and Q2(cum) determined by Simca P+12.0.1 are shown under each panel and indicate the stability and predictability of the model.</p

ESI-MS MS/MS MRM and HPLC conditions for each sphingolipid.

<p>Seg, segment; RT, retention time; Pre-ion, precursor ion; Pro-ion, product ion; CE, collision energy; ISTD, internal standard.</p>*<p>Cer(d18∶1/26∶1) and Cer(d18∶1/26∶0) were quantitated using the calibration curve of the closest counterpart when commercial standards were not available. Cer(d18∶1/26∶1) ∼Cer(d18∶1/24∶1) and Cer(d18∶1/26∶0) ∼ Cer(d18∶1/24∶0).</p

Sphingolipid biomarkers of DTH disease severity and response to triptolide treatment.

<p>The levels of seven potential sphingolipid biomarkers in the plasma (A, B) and spleen (C-G) in control, DTH model, DTH model+triptolide and control+triptolide groups. The sphingolipids were identified based on data obtained by OPLS-DA with a significant correlation (p<0.05) to ear swelling and splenic index as indicators of DTH severity. Significant differences between the two groups were evaluated by Mann-Whitney U test with the P-value shown above each plot.</p

Automatic Identification Approach for High-Performance Liquid Chromatography-Multiple Reaction Monitoring Fatty Acid Global Profiling

Fatty acids (FAs) are a group of lipid molecules that are essential to organisms. As potential biomarkers for different diseases, FAs have attracted increasing attention from both biological researchers and the pharmaceutical industry. A sensitive and accurate method for globally profiling and identifying FAs is required for biomarker discovery. The high selectivity and sensitivity of high-performance liquid chromatography-multiple reaction monitoring (HPLC-MRM) gives it great potential to fulfill the need to identify FAs from complicated matrices. This paper developed a new approach for global FA profiling and identification for HPLC-MRM FA data mining. Mathematical models for identifying FAs were simulated using the isotope-induced retention time (RT) shift (IRS) and peak area ratios between parallel isotope peaks for a series of FA standards. The FA structures were predicated using another model based on the RT and molecular weight. Fully automated FA identification software was coded using the Qt platform based on these mathematical models. Different samples were used to verify the software. A high identification efficiency (greater than 75%) was observed when 96 FA species were identified in plasma. This FAs identification strategy promises to accelerate FA research and applications