Plasma metabolomics and proteomics profiling after a postprandial challenge reveal subtle diet effects on human metabolic status

We introduce the metabolomics and proteomics based Postprandial Challenge Test (PCT) to quantify the postprandial response of multiple metabolic processes in humans in a standardized manner. The PCT comprised consumption of a standardized 500 ml dairy shake containing respectively 59, 30 and 12 energy percent lipids, carbohydrates and protein. During a 6 h time course after PCT 145 plasma metabolites, 79 proteins and 7 clinical chemistry parameters were quantified. Multiple processes related to metabolism, oxidation and inflammation reacted to the PCT, as demonstrated by changes of 106 metabolites, 31 proteins and 5 clinical chemistry parameters. The PCT was applied in a dietary intervention study to evaluate if the PCT would reveal additional metabolic changes compared to non-perturbed conditions. The study consisted of a 5-week intervention with a supplement mix of anti-inflammatory compounds in a crossover design with 36 overweight subjects. Of the 231 quantified parameters, 31 had different responses over time between treated and control groups, revealing differences in amino acid metabolism, oxidative stress, inflammation and endocrine metabolism. The results showed that the acute, short term metabolic responses to the PCT were different in subjects on the supplement mix compared to the controls. The PCT provided additional metabolic changes related to the dietary intervention not observed in non-perturbed conditions. Thus, a metabolomics based quantification of a standardized perturbation of metabolic homeostasis is more informative on metabolic status and subtle health effects induced by (dietary) interventions than quantification of the homeostatic situation

Insight in modulation of inflammation in response to diclofenac intervention: a human intervention study

Background. Chronic systemic low-grade inflammation in obese subjects is associated with health complications including cardiovascular diseases, insulin resistance and diabetes. Reducing inflammatory responses may reduce these risks. However, available markers of inflammatory status inadequately describe the complexity of metabolic responses to mild anti-inflammatory therapy. Methods. To address this limitation, we used an integrative omics approach to characterize modulation of inflammation in overweight men during an intervention with the non-steroidal anti-inflammatory drug diclofenac. Measured parameters included 80 plasma proteins, >300 plasma metabolites (lipids, free fatty acids, oxylipids and polar compounds) and an array of peripheral blood mononuclear cells (PBMC) gene expression products. These measures were submitted to multivariate and correlation analysis and were used for construction of biological response networks. Results. A panel of genes, proteins and metabolites, including PGE2 and TNF-alpha, were identified that describe a diclofenac-response network (68 genes in PBMC, 1 plasma protein and 4 plasma metabolites). Novel candidate markers of inflammatory modulation included PBMC expression of annexin A1 and caspase 8, and the arachidonic acid metabolite 5,6-DHET. Conclusion. In this study the integrated analysis of a wide range of parameters allowed the development of a network of markers responding to inflammatory modulation, thereby providing insight into the complex process of inflammation and ways to assess changes in inflammatory status associated with obesity. Trial registration. The study is registered as NCT00221052 in clinicaltrials.gov database. © 2010 van Erk et al; licensee BioMed Central Ltd

Towards harmonization of test methods for in vitro hepatic clearance studies.

Non-animal methods for toxicokinetics, such as in vitro hepatic metabolic clearance studies, play an important role in chemical risk evaluations. To gain regulatory acceptance of such clearance data, the development of a test guideline for performing in vitro hepatic clearance studies is crucial. The aim of the present study was to obtain insight in the experimental conditions of clearance studies that influence obtained intrinsic clearance (CLint) values. To that end, in vitro hepatic CLint data obtained with rat or human hepatocytes and methodological aspects of the experiments, were collected from 42 different suitable studies published between 1995 and 2018. The CLint values for the majority of chemicals differed by more than one order of magnitude. We estimated the systematic effect of different experimental setups on the CLint values using a random forest regression analysis, revealing that 'hepatocyte concentration', 'species' (rat or human hepatocytes) and 'culture medium' have the largest impact. Calculating unbound CLint (CLint,u) values slightly reduced the variation for most chemicals. Given that in vivo clearance is in general underpredicted based on in vitro clearance data, a harmonized protocol is preferably based on a protocol that provides relatively high in vitro CLint values.</p

The minipig as an alternative non-rodent model for immunogenicity testing using the TNF alpha blockers adalimumab and infliximab

Immunogenicity is a major issue of concern for monoclonal antibodies used in human diseases and is by default mainly determined in non-human primates (NHP), as target molecules are considered most similar in NHP compared to human. In this manuscript the predictive value of immunogenicity testing in minipigs for human safety is evaluated, as the immune system of the pig is functionally similar to that in other mammalian species. Adalimumab and infliximab (both monoclonal antibodies blocking TNF alpha) were used as model substances. Female Gottingen minipigs (4/group) were treated every other week with low (0.1 mg/kg), mid (1.0 mg/kg), or high dose (5 mg/kg) adalimumab or 5 mg/kg infliximab subcutaneous (SC) over a period of 8 weeks. After first and last dosing, pharmacokinetic analysis was performed. Anti-drug antibodies (ADAs) were measured on several time points. Furthermore, hematology, clinical chemistry, body weight, clinical signs, and histopathology of several organs were evaluated. No signs of toxicity of the treatments were observed in the limited organs and tissues collected. Eleven out of 12 minipigs treated with adalimumab elicited a detectable ADA response. Induction of ADA was correlated with decreased plasma levels of adalimumab. Infliximab clearance was comparable after first and last dose. Therefore, the presence of ADA directed to infliximab was considered highly unlikely. It was concluded that the minipig and NHP showed comparable suitability for immunogenicity prediction in humans. More studies with other biopharmaceutical products are needed to strengthen the status of the minipig as an alternative model for immunotoxicity testing including immunogenicit