Insights into cerumen and application in diagnostics: past, present and future prospective

Cerumen or earwax is an emerging bio-fluid in clinical diagnosis that has been very little exploited during the past decades in spite of its high diagnostic potential. It is highly abundant in diagnostic biomarkers such as genetic material, lipids, proteins, chemical elements, internal and external metabolites (e.g. hormones, volatile organic compounds, amino acids, xenobiotics etc.) reaching earwax from the blood circulation. Thus, it is able to reflect not only physiology, pathophysiology of the human body but can also detect recent and long term exposure to environmental pollutants, without the need of invasive blood tests and in the same time overcoming many disadvantages faced by using other diagnostic biological fluids. This review discusses the biology, functions, chemistry of earwax, past and current approaches for the study of its chemical composition, emphasizing how a detected variation in its composition can offer information of high clinical value, which can be useful in diagnosis of many diseases such as metabolic disorders and tumours as well as in forensic applications. It also presents details about techniques of sample collection, storage, and analysis. Moreover, it highlights concerns about the use of earwax for diagnostic purposes, which should be addressed to make earwax diagnostics a reality in the future

A population-based resource for intergenerational metabolomics analyses in pregnant women and their children: the Generation R Study

INTRODUCTION: Adverse exposures in early life may predispose children to cardio-metabolic disease in later life. Metabolomics may serve as a valuable tool to disentangle the metabolic adaptations and mechanisms that potentially underlie these associations. OBJECTIVES: T

Effect of a low glycaemic index diet during pregnancy on maternal and cord blood metabolomic profiles: results from the ROLO randomized controlled trial

Background Elevated post-prandial blood glucose during pregnancy has been associated with adverse pregnancy and offspring outcomes, such as maternal gestational diabetes and excessive foetal growth. The ROLO Study is a randomized controlled trial (RCT) investigating the effect of a low glycaemic index (GI) diet in pregnancy to prevent foetal macrosomia (birth weight > 4000 g). We described the impact of a low-GI diet on the maternal and feto-placental unit metabolism by studying how the ROLO intervention affected maternal and cord blood metabolomes. Methods Fasting maternal plasma samples pre- and post-intervention of 51 pregnant women and 132 cord blood samples were measured with a targeted metabolomics approach using liquid-chromatography coupled to tandem mass spectrometry. The differences between RCT groups were explored via multivariate models with covariates correction. Significance was set at Bonferroni-corrected level of 0.05. Results A total of 262 metabolites species, sums and ratios were investigated. While no metabolite reached statistical significance after Bonferroni correction, many maternal phospholipids and acylcarnitines were elevated in the intervention group at uncorrected 0.05 alpha level. Most species contained saturated and monounsaturated fatty acid chains with 16 or 18 carbon atoms. In cord blood, no differences were identified between RCT groups. Conclusions A low-GI diet in pregnancy was associated with a trend to modest but consistent changes in maternal lipid and fatty acid metabolism. The intervention seemed not to affect foetal metabolism. Our exploratory findings may be used to direct further investigations about low GI diets before and during pregnancy, to improve patient care for pre-conceptional and pregnant women with lipid dysregulations and potentially modulate the offspring’s risk for future metabolic diseases

Effect of a low glycaemic index diet during pregnancy on maternal and cord blood metabolomic profiles: results from the ROLO randomized controlled trial

Background Elevated post-prandial blood glucose during pregnancy has been associated with adverse pregnancy and offspring outcomes, such as maternal gestational diabetes and excessive foetal growth. The ROLO Study is a randomized controlled trial (RCT) investigating the effect of a low glycaemic index (GI) diet in pregnancy to prevent foetal macrosomia (birth weight > 4000 g). We described the impact of a low-GI diet on the maternal and feto-placental unit metabolism by studying how the ROLO intervention affected maternal and cord blood metabolomes. Methods Fasting maternal plasma samples pre- and post-intervention of 51 pregnant women and 132 cord blood samples were measured with a targeted metabolomics approach using liquid-chromatography coupled to tandem mass spectrometry. The differences between RCT groups were explored via multivariate models with covariates correction. Significance was set at Bonferroni-corrected level of 0.05. Results A total of 262 metabolites species, sums and ratios were investigated. While no metabolite reached statistical significance after Bonferroni correction, many maternal phospholipids and acylcarnitines were elevated in the intervention group at uncorrected 0.05 alpha level. Most species contained saturated and monounsaturated fatty acid chains with 16 or 18 carbon atoms. In cord blood, no differences were identified between RCT groups. Conclusions A low-GI diet in pregnancy was associated with a trend to modest but consistent changes in maternal lipid and fatty acid metabolism. The intervention seemed not to affect foetal metabolism. Our exploratory findings may be used to direct further investigations about low GI diets before and during pregnancy, to improve patient care for pre-conceptional and pregnant women with lipid dysregulations and potentially modulate the offspring’s risk for future metabolic diseases

Inhibition of mitochondrial folate metabolism drives differentiation through mTORC1-mediated purine sensing

Supporting cell proliferation through nucleotide biosynthesis is an essential requirement for cancer cells. Hence, inhibition of folate-mediated one carbon (1C) metabolism, which is required for nucleotide synthesis, has been successfully exploited in anti-cancer therapy. Here, we reveal that mitochondrial folate metabolism is upregulated in patient-derived leukaemic stem cells (LSCs). We demonstrate that inhibition of mitochondrial 1C metabolism through impairment of de novo purine synthesis has a cytostatic effect on chronic myeloid leukaemia (CML) cells. Consequently, changes in purine nucleotide levels lead to activation of AMPK signalling and suppression of mTORC1 activity. Notably, suppression of mitochondrial 1C metabolism increases expression of erythroid differentiation markers. Moreover, we find that increased differentiation occurs independently of AMPK signalling and can be reversed through reconstitution of purine levels and reactivation of mTORC1. Of clinical relevance, we identify that combination of 1C metabolism inhibition with imatinib, a frontline treatment for CML patients, decreases the number of therapy-resistant CML LSCs in a patient-derived xenograft model. Our results highlight a role for folate metabolism and purine sensing in stem cell fate decisions and leukaemogenesis

Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC

Caesarean section, but not induction of labour, is associated with major changes in cord blood metabolome

The physiology of how prelabour caesarean section (PCS) and induction of labour (IOL) in comparison to spontaneous vaginal delivery (SVD) has not been fully clarified yet. We measured 201 cord blood (CB) phospholipids and energy metabolites via LC/MS-MS in 109 newborns from the ROLO Kids study; metabolites were compared across the three parturition groups via linear mixed models with correction for multiple testing. In comparison to SVD, PCS babies had lower non-esterified fatty acids (NEFA), including sum of NEFA (p < 0.001), and trends for lower acylcarnitines. The lack of hormonal stimuli, especially catecholamines and cortisol, may underlie the metabolic changes involving gluconeogenesis from fatty acid oxidation (FAO) in PCS born infants. IOL and SVD infants showed no significant differences in metabolites, but ratios estimating carnitine palmitoyltrasferase 1 activity (precursor for FAO) were slightly higher in IOL than in SVD. Thus, IOL does not induce metabolic disadvantage when compared to SVD, though post-natal gluconeogenesis might start earlier due to the artificial solicitation in IOL. These data shed light on the physiology of parturition and may contribute to understand how mode of delivery might modulate future metabolic risks

Metabolomic Signatures in Pediatric Crohn’s Disease Patients with Mild or Quiescent Disease Treated with Partial Enteral Nutrition: A Feasibility Study

Little is known about the metabolic response of pediatric Crohn’s disease (CD) patients to partial enteral nutrition (PEN) therapy and the impact of disease activity and inflammation. We analyzed plasma samples from a nonrandomized controlled intervention study investigating the effect of partial enteral nutrition (PEN) on bone health and growth throughout one year with untargeted metabolomics using high-performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (HRMS). Thirty-four paired samples from two time points (baseline and 12 months) were analyzed. Patients (median age: 13.9 years, range: 7–18.9 years, 44% females) were in remission or had mild disease activity. The intervention group received a casein-based formula for 12 months, providing ~25% of estimated daily energy requirements. Sparse partial least squares discriminant analysis (splsda) was applied for group discrimination and identifying sources of variation to identify the impact of PEN. We also investigated the correlation of metabolites with inflammation markers, including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and fecal calprotectin. After 12 months, our results show substantial difference between PEN and non-PEN groups in the metabolome of CD patients in remission or with mild disease activity. Inflammatory markers were associated with individual compounds and chemical classes such as isoprenoids and phospholipids. Identified compounds comprise metabolites produced by human or bacterial metabolism, as well as xenobiotics recognized as flavoring agents and environmental contaminants and their biotransformation products. Further longitudinal studies that also include patients with higher disease activity are warranted to evaluate the suitability of these metabolic biomarkers for predicting disease activity

Associations of maternal and infant metabolite profiles with foetal growth and the odds of adverse birth outcomes

Background: Adaptations in maternal and foetal metabolic pathways may predispose to altered foetal growth and adverse birth outcomes. Objective: To assess the associations of maternal early-pregnancy metabolite profiles and infant metabolite profiles at birth with foetal growth from first trimester onwards and the odds of adverse birth outcomes. Methods: In a prospective population-based cohort among 976 Dutch pregnant women and their children, serum concentrations of amino acids, non-esterified fatty acids (NEFA), phospholipids (PL) and carnitines in maternal early-pregnancy blood and in cord blood were obtained by liquid-chromatography tandem mass spectrometry. Information on foetal growth was available from first trimester onwards. Results: After false discovery rate correction for multiple testing, higher infant total and individual NEFA concentrations were associated with a lower weight, length, and head circumference at birth. Higher infant total and individual acyl-lysophosphatidylcholine (lyso.PC.a) and alkyl-lysophosphatidylcholine concentrations were associated with higher weight and head circumference (lyso.PC.a only) at birth, higher odds of LGA and lower odds of SGA. Few individual maternal metabolites were associated with foetal growth measures in third trimester and at birth, but not with the odds of adverse birth outcomes. Conclusions: Our results suggest that infant metabolite profiles, particularly total and individual lyso.PC.a and NEFA concentrations, were strongly related to growth measures at birth and the odds of adverse birth outcomes. Few individual maternal early-pregnancy metabolites, but not total metabolite concentrations, are associated with foetal growth measures in third trimester and at birth