The quantification of free cholesterol (FC) and cholesteryl ester (CE) in mammalian samples is of great interest for basic science and clinical lipidomics. Here, we evaluated the feasibility of direct flow injection analysis (FIA) coupled to electrospray ionization-high resolution mass spectrometry (ESI-HRMS) to quantify FC and CE in lipid extracts from human serum, cultured cells and mouse liver. Despite poor ionization efficiency of FC, the limit of quantitation was sufficient for precise and accurate quantification of FC by multiplexed HRMS (MSX) analysis without using a derivatization step. However, it was demonstrated that upon full scan Fourier transform MS (FTMS) quantification, CE species show substantial differences in their analytical responses depending on number of double bonds, length of the acyl chain, infused lipid concentration and other lipid components. A major determinant for these response differences is their susceptibility to in-source fragmentation. In particular, introduction of double bonds lowers the degree of in-source fragmentation. Therefore, CE species-specific response factors need to be applied for CE quantification by FTMS to achieve accurate concentrations. Method validation demonstrated that FIA-ESI-HRMS (MSX and FTMS) is applicable for quantification of FC and CE in samples used in basic science as well as clinical studies such as cultured cells, tissue homogenates and serum.

See Covering the Cover synopsis on page 740.OBJECTIVE: Lipidomic changes were causally linked to metabolic diseases, but the scenario for colorectal cancer (CRC) is less clear. We investigated the CRC lipidome for putative tumor-specific alterations through analysis of 3 independent retrospective patient cohorts from 2 clinical centers, to derive a clinically useful signature. DESIGN: Quantitative comprehensive lipidomic analysis was performed using direct infusion electrospray ionization coupled with tandem mass spectrometry (ESI-MS/MS) and high-resolution mass spectrometry (HR-MS) on matched nondiseased mucosa and tumor tissue in a discovery cohort (n ¼ 106). Results were validated in 2 independent cohorts (n ¼ 28, and n ¼ 20), associated with genomic and clinical data, and lipidomic data from a genetic mouse tumor model (Apc 1638N ). RESULTS: Significant differences were found between tumor and normal tissue for glycero-, glycerophospho-, and sphingolipids in the discovery cohort. Comparison to the validation collectives unveiled that glycerophospholipids showed high interpatient variation and were strongly affected by preanalytical conditions, whereas glycero-and sphingolipids appeared more robust. Signatures of sphingomyelin and triacylglycerol (TG) species significantly differentiated cancerous from nondiseased tissue in both validation studies. Moreover, lipogenic enzymes were significantly up-regulated in CRC, and FASN gene expression was prognostically detrimental. The TG profile was significantly associated with postoperative disease-free survival and lymphovascular invasion, and was essentially conserved in murine digestive cancer, but not associated with microsatellite status, KRAS or BRAF mutations, or T-cell infiltration. CONCLUSION: Analysis of the CRC lipidome revealed a robust TG-species signature with prognostic potential. A better understanding of the cancerassociated glycerolipid and sphingolipid metabolism may lead to novel therapeutic strategies.

Pancreatic cancer has the worst prognosis among all cancers. Cancer screening of body fluids may improve the survival time prognosis of patients, who are often diagnosed too late at an incurable stage. Several studies report the dysregulation of lipid metabolism in tumor cells, suggesting that changes in the blood lipidome may accompany tumor growth. Here we show that the comprehensive mass spectrometric determination of a wide range of serum lipids reveals statistically significant differences between pancreatic cancer patients and healthy controls, as visualized by multivariate data analysis. Three phases of biomarker discovery research (discovery, qualification, and verification) are applied for 830 samples in total, which shows the dysregulation of some very long chain sphingomyelins, ceramides, and (lyso)phosphatidylcholines. The sensitivity and specificity to diagnose pancreatic cancer are over 90%, which outperforms CA 19-9, especially at an early stage, and is comparable to established diagnostic imaging methods. Furthermore, selected lipid species indicate a potential as prognostic biomarkers.

Background: The hypothesis was tested that insect meal (IM) as protein source influences intermediary metabolism of growing pigs. To test this, 30 male, 5-week-old crossbred pigs were randomly assigned to 3 groups of 10 pigs each with similar body weights (BW) and fed isonitrogenous diets either without (CON) or with 5% IM (IM5) or 10% IM (IM10) from Tenebrio molitor L. for 4 weeks and key metabolic tissues (liver, muscle, plasma) were analyzed using omics-techniques. Results: Most performance parameters did not differ across the groups, whereas ileal digestibilities of most amino acids were 6.7 to 15.6%-units lower in IM10 than in CON (P < 0.05). Transcriptomics of liver and skeletal muscle revealed a total of 166 and 198, respectively, transcripts differentially expressed between IM10 and CON (P < 0.05). Plasma metabolomics revealed higher concentrations of alanine, citrulline, glutamate, proline, serine, tyrosine and valine and a lower concentration of asparagine in IM10 than in CON (P < 0.05). Only one out of fourteen quantifiable amino acid metabolites, namely methionine sulfoxide (MetS), in plasma was elevated by 45% and 71% in IM5 and IM10, respectively, compared to CON (P < 0.05). Plasma concentrations of both, major carnitine/ acylcarnitine species and bile acids were not different across groups. Lipidomics of liver and plasma demonstrated no differences in the concentrations of triacylglycerols, cholesterol and the main phospholipids, lysophospholipids and sphingolipids between groups. The percentages of all individual phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species in the liver showed no differences between groups, except those with 6 double bonds (PC 38:6, PC 40:6, PE 38:6, PE 40:6), which were markedly lower in IM10 than in CON (P < 0.05). In line with this, the percentage of C22:6n-3 in hepatic total lipids was lower in IM10 than in the other groups (P < 0.05). Conclusions: Comprehensive analyzes of the transcriptome, lipidome and metabolome of key metabolic tissues indicate that partial or complete replacement of a conventional protein source by IM in the diet has only a weak impact on the intermediary metabolism of growing pigs. Thus, it is concluded that IM from Tenebrio molitor L. can be used as a dietary source of protein in pigs without causing adverse effects on metabolism.

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