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

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

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

Sphingolipid composition of mice liver or spleen 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 liver or spleen homogenate corresponding to 1 mg protein. Bars are expressed as means ± SD, values for each tissue sample are the average of 8 samples separately (pmol/mg protein). 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

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

Sphingolipid biomarkers found in spleen, liver, plasma and kidney.

<p>Sphingolipid biomarkers found in spleen, liver, plasma and kidney.</p

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

DTH model validation.

<p>a) The splenic index of each group. The splenic index is expressed as the ratio of the weight of the spleen weight to the weight of the mouse. The four groups include: control, DTH model, DTH model+triptolide and control+triptolide, each of which contains eight samples. Statistical differences detected between the control & DTH model groups and the DTH model & DTH model+triptolide groups are indicated by the P-value between each group. b) Ear swelling in each group. Ear swelling was expressed as the difference between the weight of left and right ear patches obtained from 8 mm punches 30 h after challenge. The punches were obtained in a blinded manner. Data are expressed as the mean ± SD, and the values for each sample are the mean of eight separate samples.</p