Kit-based assays, such as AbsoluteIDQTM p150, are widely used in large cohort studies and provide a standardized method to quantify blood concentrations of phosphatidylcholines (PCs). Many disease-relevant associations of PCs were reported using this method. However, their interpretation is hampered by lack of functionally-relevant information on the detailed fatty acid side-chain compositions as only the total number of carbon atoms and double bonds is identified by the kit. To enable more substantiated interpretations, we characterized these PC sums using the side-chain resolving LipidyzerTM platform, analyzing 223 samples in parallel to the AbsoluteIDQTM. Combining these datasets, we estimated the quantitative composition of PC sums and subsequently tested their replication in an independent cohort. We identified major constituents of 28 PC sums, revealing also various unexpected compositions. As an example, PC 16:0_22:5 accounted for more than 50% of the PC sum with in total 38 carbon atoms and 5 double bonds (PC aa 38:5). For 13 PC sums, we found relatively high abundances of odd-chain fatty acids. In conclusion, our study provides insights in PC compositions in human plasma, facilitating interpretation of existing epidemiological data sets and potentially enabling imputation of PC compositions for future meta-analyses of lipidomics data.

Angiotensin-I-converting enzyme (ACE) inhibitors are an important class of antihypertensives whose action on the human organism is still not fully understood. Although it is known that ACE especially cleaves COOH-terminal dipeptides from active polypeptides, the whole range of substrates and products is still unknown. When analyzing the action of ACE inhibitors, effects of genetic variation on metabolism need to be considered since genetic variance in the ACE gene locus was found to be associated with ACE-concentration in blood as well as with changes in the metabolic profiles of a general population. To investigate the interactions between genetic variance at the ACE-locus and the influence of ACE-therapy on the metabolic status we analyzed 517 metabolites in 1,361 participants from the KORA F4 study. We replicated our results in 1,964 individuals from TwinsUK. We observed differences in the concentration of five dipeptides and three ratios of di- and oligopeptides between ACE inhibitor users and non-users that were genotype dependent. Such changes in the concentration affected major homozygotes, and to a lesser extent heterozygotes, while minor homozygotes showed no or only small changes in the metabolite status. Two of these resulting dipeptides, namely aspartylphenylalanine and phenylalanylserine, showed significant associations with blood pressure which qualifies them—and perhaps also the other dipeptides—as readouts of ACE-activity. Since so far ACE activity measurement is substrate specific due to the usage of only one oligopeptide, taking several dipeptides as potential products of ACE into account may provide a broader picture of the ACE activity.

Identification of metabolites in non-targeted metabolomics continues to be a bottleneck in metabolomics studies in large human cohorts. Unidentified metabolites frequently emerge in the results of association studies linking metabolite levels to, for example, clinical phenotypes. For further analyses these unknown metabolites must be identified. Current approaches utilize chemical information, such as spectral details and fragmentation characteristics to determine components of unknown metabolites. Here, we propose a systems biology model exploiting the internal correlation structure of metabolite levels in combination with existing biochemical and genetic information to characterize properties of unknown molecules. Levels of 758 metabolites (439 known, 319 unknown) in human blood samples of 2279 subjects were measured using a non-targeted metabolomics platform (LC-MS and GC-MS). We reconstructed the structure of biochemical pathways that are imprinted in these metabolomics data by building an empirical network model based on 1040 significant partial correlations between metabolites. We further added associations of these metabolites to 134 genes from genome-wide association studies as well as reactions and functional relations to genes from the public database Recon 2 to the network model. From the local neighborhood in the network, we were able to predict the pathway annotation of 180 unknown metabolites. Furthermore, we classified 100 pairs of known and unknown and 45 pairs of unknown metabolites to 21 types of reactions based on their mass differences. As a proof of concept, we then looked further into the special case of predicted dehydrogenation reactions leading us to the selection of 39 candidate molecules for 5 unknown metabolites. Finally, we could verify 2 of those candidates by applying LC-MS analyses of commercially available candidate substances. The formerly unknown metabolites X-13891 and X-13069 were shown to be 2-dodecendioic acid and 9-tetradecenoic acid, respectively. Our data-driven approach based on measured metabolite levels and genetic associations as well as information from public resources can be used alone or together with methods utilizing spectral patterns as a complementary, automated and powerful method to characterize unknown metabolites.

Running title: Composition of phosphatidylcholine sums in human plasmaAbbreviations: CV, coefficient of variation; conc., concentration; mGWAS, genome-wide association study with metabolic traits, MWAS, metabolome-wide association study, OGTT, oral glucose tolerance test; OLTT, oral lipid tolerance test; PC, phosphatidylcholine; pv, p-value; R, rest that consists of the totality of nonmeasured single PCs, SD, standard deviation 3 Abstract Kit-based assays, such as AbsoluteIDQ TM p150, are widely used in large cohort studies and provide a standardized method to quantify blood concentrations of phosphatidylcholines (PCs). Many disease-relevant associations of PCs were reported using this method. However, their interpretation is hampered by lack of functionally relevant information on the detailed fatty acid side chain compositions as only the total number of carbon atoms and double bonds is identified by the kit. To enable more substantiated interpretations, we characterized these PC sums using the side chain resolving Lipidyzer TM platform, analyzing 223 samples in parallel to the AbsoluteIDQ TM . Combining these datasets, we estimated the quantitative composition of PC sums and subsequently tested their replication in an independent cohort. We identified major constituents of 28 PC sums, revealing also various unexpected compositions. As an example, PC 16:0_22:5 accounted for more than 50% of the PC sum with in total 38 carbon atoms and 5 double bonds (PC aa 38:5). For 13 PC sums, we found relatively high abundances of odd-chain fatty acids. In conclusion, our study provides insights in PC compositions in human plasma, facilitating interpretation of existing epidemiological datasets and potentially enabling imputation of PC compositions for future meta-analyses of lipidomics data.

Supplementary data are available at Bioinformatics online.