Objective: Metabolomics measurements of eccrine sweat may provide novel and relevant biomedical information to support predictive, preventive and personalised medicine (3PM). However, only limited data is available regarding metabolic alterations accompanying chemotherapy of breast cancer patients related to residual cancer burden (RCB) or therapy response. Here, we have applied Metabo-Tip, a non-invasive metabolomics assay based on the analysis of eccrine sweat from the fingertips, to investigate the feasibility of such an approach, especially with respect to drug monitoring, assessing lifestyle parameters and stratification of breast cancer patients. Methods: Eccrine sweat samples were collected from breast cancer patients (n = 9) during the first cycle of neoadjuvant chemotherapy at four time points in this proof-of-concept study at a Tertiary University Hospital. Metabolites in eccrine sweat were analysed using mass spectrometry. Blood plasma samples from the same timepoints were also collected and analysed using a validated targeted metabolomics kit, in addition to proteomics and fatty acids/oxylipin analysis. Results: A total of 247 exogenous small molecules and endogenous metabolites were identified in eccrine sweat of the breast cancer patients. Cyclophosphamide and ondansetron were successfully detected and monitored in eccrine sweat of individual patients and accurately reflected the administration schedule. The non-essential amino acids asparagine, serine and proline, as well as ornithine were significantly regulated in eccrine sweat and blood plasma over the therapy cycle. However, their distinct time-dependent profiles indicated compartment-specific distributions. Indeed, the metabolite composition of eccrine sweat seems to largely resemble the composition of the interstitial fluid. Plasma proteins and fatty acids/oxylipins were not affected by the first treatment cycle. Individual smoking habit was revealed by the simultaneous detection of nicotine and its primary metabolite cotinine in eccrine sweat. Stratification according to RCB revealed pronounced differences in the metabolic composition of eccrine sweat in these patients at baseline, e.g., essential amino acids, possibly due to the systemic contribution of breast cancer and its impact on metabolic turnover. Conclusion: Mass spectrometry-based analysis of metabolites from eccrine sweat of breast cancer patients successfully qualified lifestyle parameters for risk assessment and allowed us to monitor drug treatment and systemic response to therapy. Moreover, eccrine sweat revealed a potentially predictive metabolic pattern stratifying patients by the extent of the metabolic activity of breast cancer tissue at baseline. Eccrine sweat is derived from the otherwise hardly accessible interstitial fluid and, thus, opens up a new dimension for biomonitoring of breast cancer in secondary and tertiary care. The simple sample collection without the need for trained personnel could also enable decentralised long-term biomonitoring to assess stable disease or disease progression. Eccrine sweat analysis may indeed significantly advance 3PM for the benefit of breast cancer patients.

Metabolomics is an emerging and powerful molecular profiling method supporting clinical investigations. For clinical metabolomics studies, serum is commonly used. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may proof relevant as platelet counts and function may vary substantially in individuals. Applying a multi-omics analysis strategy comprising proteins and metabolites with a focus on lipid mediators, we systematically investigated serum and plasma obtained from the same healthy donors. While Biocrates MxP Quant 500 results correlated well (n=461, R ² =0.991), lipid mediators (n=77, R ² =0.906) and proteins (n=322, R ² =0.860) differed substantially between serum and plasma. Actually, secretome analysis of activated platelets identified all proteins and most lipid mediators significantly enriched in serum when compared to plasma. Furthermore, a prospective, randomized, controlled parallel group metabolomics trial was performed, monitored by serum and plasma analyses. Healthy individuals received either acetylsalicylic acid, affecting platelets, or omega-3 fatty acids, hardly affecting platelets, for a period of seven days. In the acetylsalicylic acid group, serum analysis apparently demonstrated a significant drug-induced downregulation of the lipid mediators TXB2 and 12-HETE. The absence of these observation in plasma analyses suggested that these drug effects took place only during blood coagulation. Other effects of acetylsalicylic acid on alpha-linolenic acid and the fatty acid composition of triglycerides were detected both in serum and plasma. In the omega-3 fatty acid group, serum and plasma analysis results did not differ. These data strongly support the hypothesis that the serum metabolome is substantially confounded by platelets. Key points: Serum metabolomics data are confounded by platelets Clinical evaluation of drug effects should be based on plasma metabolomics

Peroxisomes are subcellular compartments that host a variety of metabolic pathways, including the chain shortening of fatty acids (FAs) by beta-oxidation and certain steps in the formation of ether lipids. Here, we describe the development of a GC-MS/MS-based method for the simultaneous and reproducible determination of key metabolites of these pathways, also including less common FA species related to peroxisomal metabolism that are typically not part of standard analytical methods. We for the first time utilize 1-chlorobutane for the extraction of FAs as an effective alternative to commonly used extraction solvents. 1-Chlorobutane offers a broader polarity range than hexane and lower toxicity relative to chloroform with solvent consumption of less than one mL per sample. Six saturated long to very long-chain FAs, nine polyunsaturated FAs (PUFAs), two dicarboxylic FAs and 1-O-octadecyl glycerol (ODG, batyl alcohol) were extracted simultaneously. The method was validated using fibroblasts and for the majority of FA species accuracies ranged from 80 to 110 % with precision values (CV %) from 6 to 20 %. The measurement of ODG is for the first time described as marker for the estimation of the cellular ether lipid synthesis rate. The suitability of the method was demonstrated by the analysis of primary human fibroblasts from controls and individuals with peroxisomal disorders. This cell type represents a widely used model system for the investigation of peroxisomal metabolism and disease, thus rendering our protocol a valuable addition to the toolkit for studying peroxisomal pathways.

Background: Obesity in pregnancy is linked to adverse clinical outcomes such as gestational diabetes. Recently, a risk score calculated by different ceramide concentrations was recognized as a new way to investigate cardiovascular risk. The aim was to analyze if the ceramide risk score and cardiometabolic risk vary between normal-weight, obese, and females with prior Roux-en-Y bypass surgery (RYGB) during pregnancy. Methods: Three cohorts were investigated: first, 25 pregnant females with a history of RYGB; second, 19 with preconception BMI ≥ 35 kg/m²; and third, 19 normal-weight (preconception BMI < 25 kg/m²). Around the 24th to 28th weeks of gestation routine laboratory assessments, 3 h 75 g oral and intravenous glucose tolerance tests were carried out. The correlation of ceramide risk scores and ceramide ratios (Cer(d18:1/18:0)/Cer(d18:1/16:0)) with metabolic parameters was analyzed via Pearson correlation. The cohorts were compared via ANOVA and unpaired t-tests. Results: The RYGB cohort had lower ceramide risk scores and ratios compared to obese pregnant females (7.42 vs. 9.34, p = 0.025; 0.33 vs. 0.47, p < 0.001). Ceramide risk score and ratio were found to correlate negatively with insulin sensitivity (measured with the Matsuda (r = −0.376, p = 0.031; r = −0.455, p = 0.008) and calculated sensitivity index (r = −0.358, p = 0.044; r = −0.621, p < 0.001) in females without RYGB. The ceramide risk score correlated positively with body fat in RYGB females (r = 0.650, p = 0.012). Conclusions: We found that females after RYGB have lower ceramide risk scores and ceramide ratios compared to obese pregnant females, possibly indicating lower metabolic risk.

Peroxisomes are subcellular compartments that host a variety of metabolic pathways, including the chain shortening of fatty acids (FAs) by beta-oxidation and certain steps in the formation of ether lipids. Here, we describe the development of a GC-MS/MS-based method for the simple simultaneous and reliable determination of key metabolites of these pathways, also including less common FA species related to peroxisomal metabolism that are typically not part of standard analytical methods. We for the first time utilize 1-chlorobutane for the extraction of FAs as an effective alternative to commonly used extraction solvents. 1-chlorobutane offers a broader polarity range than hexane and lower toxicity relative to chloroform with solvent consumption of less than 1 mL per sample. Six saturated long to very long-chain FAs, nine polyunsaturated FAs (PUFAs), two dicarboxylic FAs and 1-O-octadecyl glycerol (ODG, batyl alcohol) were extracted simultaneously. The method was validated using fibroblasts and for the majority of FA species accuracies ranged from 80 to 110% with coefficients of variations (CVs) from 6 to 20 %. For the estimation of the ether lipid synthesis rate the measurement of ODG was established as reliable marker. The suitability of the method was demonstrated by the analysis of primary human fibroblasts from controls and individuals with peroxisomal disorders. This cell type represents a commonly used model system for the investigation of peroxisomal metabolism and disease, thus rendering our protocol a valuable addition to the toolkit for studying peroxisomal pathways.

Isotope tracing experiments in cellular metabolomics are challenged by the multiple isomers and in-source fragments, which need to be considered to obtain unbiased isotopologue ratio measurements. Thus, both, selectivity and sensitivity are key requirements for customized workflows. Trapped ion mobility spectrometry (TIMS) introduces an additional separation dimension to mass spectrometry, separating otherwise co-eluting isomers by measuring the ion mobility of a molecule. This study shows for the first time, the potential of this MS platform for accurate isotopologue assessment as showcased in isotope tracer experiments using mammalian cells. The validation exercise focused on spectral accuracy, precision, and metabolite detection capabilities and comprised independent measurements on an orbitrap-based platform. Hydrophilic interaction chromatography, in combination with TIMS-TOF-MS delivered excellent results, with a minimum trueness bias and excellent precision (CV%) between 0.3 % and 6.4 %. The ion mobility separation allowed for differentiation of the otherwise co-eluting isomers fructose-6-phosphate (F6P) and glucose-1-phosphate (G1P). Overall, isotopologue distributions were in good agreement upon crossvalidation with the orbitrap platform. Finally, a proof-of-concept tracer study addressed the activity of the glycolysis and the pentose phosphate pathway (PPP) in resting and endotoxin activated macrophages. We confirmed an activation of glycolysis and PPP in LPS activated macrophages, but found a potentially reduced relative contribution of glucose-6-phosphate (G6P) to increased F6P pools. Our findings imply that TIMS is a powerful technology for the reliable measurements of isotope distribution analysis in metabolic tracing experiments. By implementation of ion mobility, it is now possible to generate distinct isotopologue patterns for G1P and F6P in isotope tracer experiments. F6P plays a crucial role in glycolysis and PPP, highlighting the importance of precise analytical measurements. This is particularly true for metabolic studies in immunology and cancer research.