Absoluteidq p180

Metabolic profiling was performed on plasma samples in the batch mode by using the Waters XEVO TQ MS mass spectrometry system (Waters Limited, Mississauga, Ontario, Canada). Samples together with the Quality Control material at three concentration levels and calibrators were extracted, internal standard added and data question files exported for data processing. The analysis and data processing were performed with the help of the commercial reagent kit Biocrates AbsoluteIDQ p180. The kit allows quantitative analysis of a metabolic panel consisting of 186 metabolites; these included 90 species of glycerophospholipids, 40 species acylcarnitines (including free L-carnitine), 21 species of amino acids, 19 species biogenic amines, 15 species of sphingolipids and one hexose of which 90% was glucose. Additional details on the reagent kit, data acquisition and processing were previously described [15 ].

Metabolites in plasma samples were determined using a commercially available kit (AbsoluteIDQ p180; BIOCRATES Life Sciences AG, Innsbruck, Austria). Briefly, an aliquot of 10 μL plasma was prepared and processed according to the manufacturer’s instructions as previously described [41 (link),42 (link)]. Biogenic amines were measured using the ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and lipid species were quantified by flow injection analysis (FIA) coupled with MS/MS. Metabolite concentrations were calculated and expressed in μM. The global analysis of plasma metabolites was performed as described elsewhere [43 (link)].

For all 388 women in the clinical cohort, blood samples were collected as part of the NOWAC Post-genome cohort as described previously [10 (link)]. In a subset of these samples, i.e. approximately 100 samples, a series of omics analyses have already been carried out. For details on the laboratory methods, please refer to S1 Appendix. Briefly, mRNA gene expression profiles were analyzed from PAXgene blood RNA samples by Illumina HumanHT-12 Expression BeadChip microarrays (Illumina, Inc. San Diego, CA, USA). The analyses were carried out by a certified Illuimna service provider, the Genomics Core Facility (GCF), Norwegian University of Science and Technology, Trondheim, Norway. For plasma metabolomics, a liquid chromatography-mass spectrometry (LC-MS/MS) based kit “AbsoluteIDQ p180” (Biocrates Life Sciences, Innsbruck, Austria) was used for quantification of up to 188 metabolites. The Swedish Metabolomics Centre in Umeå, Sweden carried out the analyses. Extraction and profiling of miRNA in plasma were performed by Exiqon Services (Vedbaek, Denmark). Total RNA was extracted using the mirCURY^™ RNA isolation kit, and a PCR-based panel of 372 probes was used for miRNA profiling (miRCURY LNA^™ Universal RT microRNA PCR Human panel I, Qiagen, Hilden, Germany). See S1 Appendix for detailed information on the analyses of blood samples.

As summarized in Table 1, pre-diagnostic blood samples were assayed at the Helmholtz Zentrum (München, Germany) for the second colorectal cancer study, at Imperial College London (UK) for the endometrial cancer study, and at IARC for all other studies. Data for a total of 171 metabolites were acquired by tandem mass spectrometry using either the AbsoluteIDQ p150 (for the second colorectal cancer study) or the AbsoluteIDQ p180 commercial kit (Biocrates Life Science AG, Innsbruck Austria). Two successive assays were used, liquid chromatography-tandem mass spectrometry (LC-MS/MS) for amino acids and biogenic amines, and flow injection analysis-tandem mass spectrometry (FIA-MS/MS) for the other metabolites. Samples were either serum or citrate plasma, and samples within each study were all from the same type of blood matrix, except for the breast cancer study (Table 1). Samples of each case-control pair were assayed on the same batch (and in the same laboratory).

Before exclusions of hormone users, a total of 3179 samples were available for 3163 women. All samples, plasma (in 95.1% of samples) or serum, were assayed by liquid chromatography-mass spectrometry using the AbsoluteIDQ p180 commercial kit (Biocrates Life Sciences AG, Innsbruck, Austria). A total of 2289 (72.0%) samples were assayed at the laboratory of the Biomarkers Group at IARC (breast, colorectal, kidney, and liver cancer studies); 851 (26.8%) at the Imperial College, London; and 39 (1.2%) at the Helmholtz Zentrum, München, Germany. At IARC, analyses were run on a QTRAP5500 (breast, kidney, and liver cancer studies) and TQ4500 (colorectal cancer study) mass spectrometers (AB Sciex, Framingham, MA, USA), while at the Imperial College London and Helmholtz Zentrum, analyses were run using an API4000TQ (endometrial and gallbladder cancer studies). All analyses for a given study were performed using the same instrument. Sixteen participants had their samples analyzed in two different studies, at IARC and at the Helmholtz Zentrum, for whom the metabolite concentrations were averaged over the two measures.
Out of the 3179 samples, arginine concentrations could not be quantified in five, as they were below the lower limit of quantification (LLOQ) and were therefore imputed to half this LLOQ, consistently with previous work [1 (link)].