Metabolomics of dietary fatty acid restriction in patients with phenylketonuria

Patients with phenylketonuria (PKU) have to follow a lifelong phenylalanine restricted diet. This type of diet markedly reduces the intake of saturated and unsaturated fatty acids especially long chain polyunsaturated fatty acids (LC-PUFA). Long-chain saturated fatty acids are substrates of mitochondrial fatty acid oxidation for acetyl-CoA production. LC-PUFA are discussed to affect inflammatory and haemostaseological processes in health and disease. The influence of the long term PKU diet on fatty acid metabolism with a special focus on platelet eicosanoid metabolism has been investigated in the study presented here. 12 children with PKU under good metabolic control and 8 healthy controls were included. Activated fatty acids (acylcarnitines C6-C18) in dried blood and the cholesterol metabolism in serum were analyzed by liquid chromatographic tandem mass spectrometry (LC-MS/MS). Fatty acid composition of plasma glycerophospholipids was determined by gas chromatography. LC-PUFA metabolites were analyzed in supernatants by LC-MS/MS before and after platelet activation and aggregation using a standardized protocol. Patients with PKU had significantly lower free carnitine and lower activated fatty acids in dried blood compared to controls. Phytosterols as marker of cholesterol (re-) absorption were not influenced by the dietary fatty acid restriction. Fatty acid composition in glycerophospholipids was comparable to that of healthy controls. However, patients with PKU showed significantly increased concentrations of y-linolenic acid (C18:3n-6) a precursor of arachidonic acid. In the PKU patients significantly higher platelet counts were observed. After activation with collagen platelet aggregation and thromboxane B(2) and thromboxane B(3) release did not differ from that of healthy controls. Long-term dietary fatty acid restriction influenced the intermediates of mitochondrial beta-oxidation. No functional influence on unsaturated fatty acid metabolism and platelet aggregation in patients with PKU was detected

Serum amino acid profiles and their alterations in colorectal cancer

Mass spectrometry-based serum metabolic profiling is a promising tool to analyse complex cancer associated metabolic alterations, which may broaden our pathophysiological understanding of the disease and may function as a source of new cancer-associated biomarkers. Highly standardized serum samples of patients suffering from colon cancer (n=59) and controls (n=58) were collected at the University Hospital Leipzig. We based our investigations on amino acid screening profiles using electrospray tandem-mass spectrometry. Metabolic profiles were evaluated using the Analyst 1.4.2 software. General, comparative and equivalence statistics were performed by R 2.12.2. 11 out of 26 serum amino acid concentrations were significantly different between colorectal cancer patients and healthy controls. We found a model including CEA, glycine, and tyrosine as best discriminating and superior to CEA alone with an AUROC of 0.878 (95% CI 0.815-0.941). Our serum metabolic profiling in colon cancer revealed multiple significant disease-associated alterations in the amino acid profile with promising diagnostic power. Further large-scale studies are necessary to elucidate the potential of our model also to discriminate between cancer and potential differential diagnoses. In conclusion, serum glycine and tyrosine in combination with CEA are superior to CEA for the discrimination between colorectal cancer patients and control

Pancreatic carcinoma, pancreatitis, and healthy controls: metabolite models in a three-class diagnostic dilemma

Metabolomics as one of the most rapidly growing technologies in the "-omics” field denotes the comprehensive analysis of low molecular-weight compounds and their pathways. Cancer-specific alterations of the metabolome can be detected by high-throughput mass-spectrometric metabolite profiling and serve as a considerable source of new markers for the early differentiation of malignant diseases as well as their distinction from benign states. However, a comprehensive framework for the statistical evaluation of marker panels in a multi-class setting has not yet been established. We collected serum samples of 40 pancreatic carcinoma patients, 40 controls, and 23 pancreatitis patients according to standard protocols and generated amino acid profiles by routine mass-spectrometry. In an intrinsic three-class bioinformatic approach we compared these profiles, evaluated their selectivity and computed multi-marker panels combined with the conventional tumor marker CA19-9. Additionally, we tested for non-inferiority and superiority to determine the diagnostic surplus value of our multi-metabolite marker panels. Compared to CA19-9 alone, the combined amino acid-based metabolite panel had a superior selectivity for the discrimination of healthy controls, pancreatitis, and pancreatic carcinoma patients $$[ {\text{volume under ROC surface}}\;\left( {\text{VUS}} \right) = 0. 8 9 1 { }\left( { 9 5\,\% {\text{ CI }}0. 7 9 4- 0. 9 6 8} \right)].$$ We combined highly standardized samples, a three-class study design, a high-throughput mass-spectrometric technique, and a comprehensive bioinformatic framework to identify metabolite panels selective for all three groups in a single approach. Our results suggest that metabolomic profiling necessitates appropriate evaluation strategies and—despite all its current limitations—can deliver marker panels with high selectivity even in multi-class setting

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in ‘functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP

Impact of Myeloperoxidase-derived oxidants on the product profile of human 5-Lipoxygenase

Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl– system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils

Targeted Lipidomics for Characterization of PUFAs and Eicosanoids in Extracellular Vesicles

Lipids are increasingly recognized as bioactive mediators of extracellular vesicle (EV) functions. However, while EV proteins and nucleic acids are well described, EV lipids are insufficiently understood due to lack of adequate quantitative methods. We adapted an established targeted and quantitative mass spectrometry (LC-MS/MS) method originally developed for analysis of 94 eicosanoids and seven polyunsaturated fatty acids (PUFA) in human plasma. Additionally, the influence of freeze–thaw (FT) cycles, injection volume, and extraction solvent were investigated. The modified protocol was applied to lipidomic analysis of differently polarized macrophage-derived EVs. We successfully quantified three PUFAs and eight eicosanoids within EVs. Lipid extraction showed reproducible PUFA and eicosanoid patterns. We found a particularly high impact of FT cycles on EV lipid profiles, with significant reductions of up to 70%. Thus, repeated FT will markedly influence analytical results and may alter EV functions, emphasizing the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs. EV lipid profiles differed largely depending on the polarization of the originating macrophages. Particularly, we observed major changes in the arachidonic acid pathway. We emphasize the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs

Potential of Dried Blood Self-Sampling for Cyclosporine C2 Monitoring in Transplant Outpatients

Background. Close therapeutic drug monitoring of Cyclosporine (CsA) in transplant outpatients is a favourable procedure to maintain the long-term blood drug levels within their respective narrow therapeutic ranges. Compared to basal levels (C0), CsA peak levels (C2) are more predictive for transplant rejection. However, the application of C2 levels is hampered by the precise time of blood sampling and the need of qualified personnel. Therefore, we evaluated a new C2 self-obtained blood sampling in transplant outpatients using dried capillary and venous blood samples and compared the CsA levels, stability, and clinical practicability of the different procedures. Methods. 55 solid organ transplant recipients were instructed to use single-handed sampling of each 50 μL capillary blood and dried blood spots by finger prick using standard finger prick devices. We used standardized EDTA-coated capillary blood collection systems and standardized filter paper WS 903. CsA was determined by LC-MS/MS. The patients and technicians also answered a questionnaire on the procedure and sample quality. Results. The C0 and C2 levels from capillary blood collection systems (C0 [ng/mL]: 114.5 ± 44.5; C2: 578.2 ± 222.2) and capillary dried blood (C0 [ng/mL]: 175.4 ± 137.7; C2: 743.1 ± 368.1) significantly (P < .01) correlated with the drug levels of the venous blood samples (C0 [ng/mL]: 97.8 ± 37.4; C2: 511.2 ± 201.5). The correlation at C0 was ρcap.-ven. = 0.749, and ρdried blood-ven = 0.432; at C2: ρcap.-ven. = 0.861 and ρdried blood-ven = 0.711. The patients preferred the dried blood sampling because of the more simple and less painful procedure. Additionally, the sample quality of self-obtained dried blood spots for LC-MS/MS analytics was superior to the respective capillary blood samples. Conclusions. C2 self-obtained dried blood sampling can easily be performed by transplant outpatients and is therefore suitable and cost-effective for close therapeutic drug monitoring

Development of a rapid and specific MALDI-TOF mass spectrometric assay for SARS-CoV-2 detection

We have developed a rapid and highly specific assay for detecting and monitoring SARS-CoV-2 infections by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). As MALDI-TOF mass spectrometers are available in a clinical setting, our assay has the potential to serve as alternative to the commonly used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Sample preparation prior to MALDI-TOF-MS involves the tryptic digestion of SARS-CoV-2 proteins, followed by an enrichment of virus-specific peptides from SARS-CoV-2 nucleoprotein via magnetic antibody beads. Our MALDI-TOF-MS method allows the detection of SARS-CoV-2 nucleoprotein in sample collection medium as low as 8 amol/µl. MALDI-TOF mass spectra are obtained in just a few seconds, which makes our MS-based assay suitable for a high-throughput screening of SARS-CoV-2 in healthcare facilities in addition to PCR. Due to the specific detection of virus peptides, different SARS-CoV-2 variants are readily distinguished from each other. Specifically, we show that our MALDI-TOF-MS assay discriminates SARS-CoV-2 strain B.1.617.2 “delta variant” from all other variants in patients’ samples, making our method highly valuable to monitor the emergence of new virus variants

Evaluation of five multisteroid LC‒MS/MS methods used for routine clinical analysis: comparable performance was obtained for nine analytes

Objectives: A mass spectrometry (LC‒MS/MS)-based interlaboratory comparison study was performed for nine steroid analytes with five participating laboratories. The sample set contained 40 pooled samples of human serum generated from preanalyzed leftovers. To obtain a well-balanced distribution across reference intervals of each steroid, the leftovers first underwent a targeted mixing step. Methods: All participants measured a sample set once using their own multianalyte protocols and calibrators. Four participants used in-house developed measurement platforms, including IVD-CE certified calibrators, which were used by three participants; the 5th lab used the whole LC‒MS kit from an IVD manufacturer. All labs reported results for 17-hydroxyprogesterone, androstenedione, cortisol, and testosterone, and four labs reported results for 11-deoxycortisol, corticosterone, cortisone, dehydroepiandrosterone sulfate (DHEAS), and progesterone. Results: Good or acceptable overall comparability was found in Bland‒Altmann and Passing‒Bablok analyses. Mean bias against the overall mean remained less than ±10 % except for DHEAS, androstenedione, and progesterone at one site and for cortisol and corticosterone at two sites (max. -18.9 % for androstenedione). The main analytical problems unraveled by this study included a bias not previously identified in proficiency testing, operator errors, non-supported matrix types and higher inaccuracy and imprecision at lower ends of measuring intervals. Conclusions: This study shows that intermethod comparison is essential for monitoring the validity of an assay and should serve as an example of how external quality assessment could work in addition to organized proficiency testing schemes

Folate and Cobalamin Serum Levels in Healthy Children and Adolescents and Their Association with Age, Sex, BMI and Socioeconomic Status

This study proposes age- and sex-specific percentiles for serum cobalamin and folate, and analyzes the effects of sex, age, body mass index (BMI), and socioeconomic status (SES) on cobalamin and folate concentrations in healthy children and adolescents. In total, 4478 serum samples provided by healthy participants (2 months–18.0 years) in the LIFE (Leipzig Research Centre for Civilization Diseases) Child population-based cohort study between 2011 and 2015 were analyzed by electrochemiluminescence immunoassay (ECLIA). Continuous age-and sex-related percentiles (2.5th, 10th, 50th, 90th, 97.5th) were estimated, applying Cole’s LMS method. In both sexes, folate concentrations decreased continuously with age, whereas cobalamin concentration peaked between three and seven years of age and declined thereafter. Female sex was associated with higher concentrations of both vitamins in 13- to 18-year-olds and with higher folate levels in one- to five-year-olds. BMI was inversely correlated with concentrations of both vitamins, whilst SES positively affected folate but not cobalamin concentrations. To conclude, in the assessment of cobalamin and folate status, the age- and sex-dependent dynamic of the respective serum concentrations must be considered. While BMI is a determinant of both vitamin concentrations, SES is only associated with folate concentrations