Murine deficiency of peroxisomal L-bifunctional protein (EHHADH) causes medium-chain 3-hydroxydicarboxylic aciduria and perturbs hepatic cholesterol homeostasis.

Peroxisomes play an essential role in the ß-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA ß-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA ß-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA ß-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA ß-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA ß-oxidation is a regulator of hepatic cholesterol biosynthesis.

Measurement of bempedoic acid and its keto metabolite in human plasma and urine using solid phase extraction and electrospray LC-MS/MS.

Bempedoic acid, a new therapeutic for treatment of hypercholesterolemia, inhibits hepatic ATP-citrate lyase in the cholesterol synthesis pathway after its conjugation with coenzyme A. Sensitive and selective methods were required to study the pharmacokinetic behavior of bempedoic acid and its active 8-keto metabolite in clinical studies. A mixed mode anion exchange extraction on 96-well plates was developed to favor high, selective recoveries of these dicarboxylic acids from urine or plasma. Adsorptive losses in urine led to inaccurate measurements unless samples were acidified and diluted with isopropanol prior to any specimen transfers. Tandem mass spectrometry with negative ion electrospray ionization permitted lower limits of measurement of 20 and 10 ng/mL for the drug and metabolite in either matrix. The methods were validated to current regulatory standards and have been the basis for pharmacokinetic measurements in 26 clinical studies involving over 15,000 samples.

Urine dicarboxylic acids change in pre-symptomatic Alzheimer's disease and reflect loss of energy capacity and hippocampal volume.

Non-invasive biomarkers will enable widespread screening and early diagnosis of Alzheimer's disease (AD). We hypothesized that the considerable loss of brain tissue in AD will result in detection of brain lipid components in urine, and that these will change in concert with CSF and brain biomarkers of AD. We examined urine dicarboxylic acids (DCA) of carbon length 3-10 to reflect products of oxidative damage and energy generation or balance that may account for changes in brain function in AD. Mean C4-C5 DCAs were lower and mean C7-C10 DCAs were higher in the urine from AD compared to cognitively healthy (CH) individuals. Moreover, mean C4-C5 DCAs were lower and mean C7-C9 were higher in urine from CH individuals with abnormal compared to normal CSF amyloid and Tau levels; i.e., the apparent urine changes in AD also appeared to be present in CH individuals that have CSF risk factors of early AD pathology. In examining the relationship between urine DCAs and AD biomarkers, we found short chain DCAs positively correlated with CSF Aß42, while C7-C10 DCAs negatively correlated with CSF Aß42 and positively correlated with CSF Tau levels. Furthermore, we found a negative correlation of C7-C10 DCAs with hippocampal volume (p < 0.01), which was not found in the occipital volume. Urine measures of DCAs have an 82% ability to predict cognitively healthy participants with normal CSF amyloid/Tau. These data suggest that urine measures of increased lipoxidation and dysfunctional energy balance reflect early AD pathology from brain and CSF biomarkers. Measures of urine DCAs may contribute to personalized healthcare by indicating AD pathology and may be utilized to explore population wellness or monitor the efficacy of therapies in clinical trials.

Non-invasive urinary metabolomics reveals metabolic profiling of polycystic ovary syndrome and its subtypes.

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder, which affects 4-10 % women of reproductive age. Though accumulating scientific evidence, its pathogenesis remains unclear. In the current study, metabolic profiling as well as diagnostic biomarkers for different phenotypes of PCOS was investigated using non-invasive urinary GCMS based metabolomics. A total of 371 subjects were recruited for the study. They constituted the following groups: healthy women, those with hyperandrogenism (HA), women with insulin-resistance (IR) in PCOS. Two cross-comparisons with PCOS were performed to characterize metabolic disturbances. A total of 23 differential metabolites were found. The altered metabolic pathways included glyoxylate and dicarboxylate metabolism, pentose and glucuronate interconversions, and citrate cycle and butanoate metabolism. For differential diagnosis, a panel consisting of 9 biomarkers was found from the comparison of PCOS from healthy subjects. The area under the receiver operating characteristic (ROC) curve (AUC) was 0.8461 in the discovery phase. Predictive value of 89.17 % was found in the validation set. Besides, a panel of 8 biomarkers was discovered from PCOS with HA vs IR. The AUC for 8-biomarker panel was 0.8363, and a panel of clinical markers (homeostasis model assessment-insulin resistance and free androgen index) had 0.8327 in AUC. While these metabolites combined with clinical markers reached 0.9065 in AUC from the discovery phase, and 93.18 % in predictive value from the validation set. The result showed that differences of small-molecule metabolites in urine may reflect underlying pathogenesis of PCOS and serve as biomarkers for complementary diagnosis of the different phenotypes of PCOS.

Hexamoll® DINCH and DPHP metabolites in urine of children and adolescents in Germany. Human biomonitoring results of the German Environmental Survey GerES V, 2014-2017.

The production and use of the plasticisers Hexamoll® DINCH (di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate) and DPHP (di-(2-propylheptyl) phthalate) have increased after both chemicals were introduced into the market in the early 2000s as substitutes for restricted high molecular weight phthalates. During the population representative German Environmental Survey (GerES) of Children and Adolescents (GerES V, 2014-2017), we collected urine samples and measured the concentrations of DINCH and DPHP metabolites in 2228 and in a subsample of 516 participants, respectively. We detected DINCH and DPHP metabolites in 100% and 62% of the 3-17 years old children and adolescents, respectively. Geometric means of DINCH metabolites were 2.27 µg/L for OH-MINCH, 0.93 µg/L for oxo-MINCH, 1.14 µg/L for cx-MINCH and 3.47 µg/L for DINCH (Σ of OH-MINCH + cx-MINCH). Geometric means of DPHP metabolites were 0.30 µg/L for OH-MPHP, 0.32 µg/L for oxo-MPHP and 0.64 µg/L for DPHP (Σ of OH-MPHP + oxo-MPHP). The 3-5 years old children had almost 3-fold higher DINCH biomarkers levels than adolescents (14-17 years). Higher concentrations of DPHP biomarkers among young children only became apparent after creatinine adjustment. Urinary levels of DINCH but not of DPHP biomarkers were associated with the levels of the respective plasticisers in house dust. When compared to HBM health-based guidance values, we observed no exceedance of the HBM-I value of 1 mg/L for DPHP (Σ of OH-MPHP + oxo-MPHP). However, 0.04% of the children exceeded the health based guidance value HBM-I of 3 mg/L for DINCH (Σ of OH-MINCH + cx-MINCH). This finding shows that even a less toxic replacement of restricted chemicals can reach exposures in some individuals, at which, according to current knowledge, health impacts cannot be excluded with sufficient certainty. In conclusion, we provide representative data on DINCH and DPHP exposure of children and adolescents in Germany. Further surveillance is warranted to assess the substitution process of plasticisers, and to advise exposure reduction measures, especially for highly exposed children and adolescents. Providing the results to the European HBM Initiative HBM4EU will support risk assessment and risk management not only in Germany but also in Europe.

Urinary levels of phthalates and DINCH metabolites in Korean and Thai pregnant women across three trimesters.

Phthalates are anti-androgenic chemicals and may cause long-lasting adverse effects on growing fetuses. Understanding their exposure profile during pregnancy, therefore, is of public health importance. Because both behavioral and physiological changes of pregnant women are expected to be substantial, the amount of phthalate exposure is expected to vary significantly over the course of pregnancy. Temporal trend of phthalate exposure during pregnancy, however, is largely unknown, especially in Asian women. The purpose of this study is to investigate the urinary concentrations of metabolites for major phthalates and alternative plasticizers over the course of pregnancy among Korean (n = 81) and Thai women (n = 102). Twenty-four metabolites from 15 plasticizers, such as dimethyl phthalate (DMP), diethyl phthalate (DEP), di-isobutyl phthalate (DiBP), di-n-butyl phthalate (DnBP), benzyl butyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP), dioctyl phthalate (DnOP), diisononyl phthalate (DiNP), diisodecyl phthalate (DiDP), di(2-ethylhexyl) terephthalate (DEHTP), and di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate (DINCH), were measured in urine samples collected in each trimester from pregnant women. While the levels of several phthalate metabolites were significantly different by trimester among Korean women, those of Thai women were relatively consistent. Urinary metabolites of DEP and DnOP were higher in Thai pregnant women compared to Korean pregnant women. The detection frequencies of the DINCH metabolite were 67.4% and 44.9% among Korean and Thai pregnant women, respectively. However, the ratio of DINCH to DEHP metabolites was significantly higher in Thai women. According to risk assessment, 11.9% of Korean and 5.3% of Thai women were considered at risk due to phthalate exposure, and DEHP, DnBP and DiBP were identified as major risk drivers. Considering the vulnerability of growing fetuses, further studies are warranted to identify major sources of exposure to these plasticizers during pregnancy.

Severe clinical manifestation of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency associated with two novel mutations: a case report.

BACKGROUND: Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS) deficiency is an autosomal recessive inborn error of metabolism, which will give rise to failure of ketogenesis in liver during illness or fasting. It is a very rare disease with only a few patients reported worldwide, most of which had a good prognosis after proper therapies. CASE PRESENTATION: We report a 9-month-old boy with mHS deficiency presenting with unusually severe and persistent acidosis after diarrhea and reduced oral food intake. The metabolic acidosis persisted even after supplementation with sugar and alkaline solution. Blood purification and assisted respiration alleviated symptoms, but a second onset induced by respiratory infection several days later led to multiple organ failure and death. Urine organic acid analysis during the acute episode revealed a complex pattern of ketogenic dicarboxylic and 3-hydroxydicarboxylic aciduria with prominent elevation of glutaric acid and adipic acid, which seem to be specific to mHS deficiency. Plasma acylcarnitine analysis revealed elevated 3-hydroxybutyrylcarnitine and acetylcarnitine. This is the first report of elevated 3-hydroxybutyrylcarnitine in mHS deficiency. Whole exome sequencing revealed a novel compound heterozygous mutation in HMGCS2 (c.100C > T and c.1465delA). CONCLUSION: This severe case suggests the need for patients with mHS deficiency to avoid recurrent illness because it can induce severe metabolic crisis, possibly leading to death. Such patients may also require special treatment, such as blood purification. Urine organic acid profile during the acute episode may give a hint to the disease.

Time trend of exposure to the phthalate plasticizer substitute DINCH in Germany from 1999 to 2017: Biomonitoring data on young adults from the Environmental Specimen Bank (ESB).

DINCH (cyclohexane-1,2-dicarboxylic acid-diisononyl ester) is a phthalate plasticizer substitute introduced into the market in 2002. It is increasingly used especially in the production of toys, food contact materials and medical devices. In this measurement campaign on 24-h urine samples of young adults (20-29 years) from the German Environmental Specimen Bank (ESB) collected in 2010, 2011, 2013, 2015 and 2017 (in total 300 samples, 60 samples/year) we analyzed three specific, oxidized DINCH metabolites (OH-MINCH: cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester; cx-MINCH: cyclohexane-1,2-dicarboxylic acid-mono(carboxy-isooctyl) ester, oxo-MINCH: cyclohexane-1,2-dicarboxylic acid-mono(oxo-isononyl) ester). We merged these data with earlier data of the ESB from the years 1999-2012 and are now able to report levels and time trends of internal DINCH exposure from 1999 to 2017. After first detections of the major oxidized DINCH metabolite OH-MINCH in 2006 (6.7%) detection rates rapidly increased to 43.3% in 2009, 80% in 2010 and 98.3% in 2011 and 2012. From the year 2013 on we could detect OH-MINCH in every urine sample analyzed. The median concentrations of OH-MINCH rapidly increased from 0.15 µg/L in 2010 to twice the concentration in 2011 (0.31 µg/L) with further increases in 2013 (0.37 µg/L), 2015 (0.59 µg/L) and 2017 (0.70 µg/L). Similar increases, albeit at lower detection rates and concentration levels, could be observed for cx-MINCH and oxo-MINCH. All metabolites strongly correlate with each other. For the ESB study population, DINCH exposures are still far below health based guidance values such as the German Human Biomonitoring Value (HBM-I; 4,500 µg/L for the sum of OH-MINCH and cx-MINCH) or the tolerable daily intake (TDI) of EFSA (1 mg/kg bw/d). The median daily DINCH intake (DI) calculated for 2017 was 0.23 µg/kg bw/d, thus 4,310-times lower than the TDI. The maximum DI calculated for one individual in 2012 (42.60 µg/kg bw/d) was a factor of more than 20 below the TDI. The ongoing increase in DINCH exposure needs to be closely monitored in the future, including populations with potentially higher exposures such as children. This close monitoring will enable timely exposure and risk reduction measures if exposures reached critical levels, or if new toxicological data lead to lower health based guidance values. DINCH belongs to the European Human Biomonitoring Initiative (HBM4EU) priority substances for which policy relevant questions still have to be answered.

Bioavailability of phthalate and DINCH® plasticizers, after oral administration of dust to piglets.

For decades, phthalates have been widely used as plasticizers in a large number of consumer products, leading to a complex exposure to humans via ingestion, inhalation or dermal uptake. Children may have a higher unintended dust intake per day compared to adults. Therefore, dust intake of children could pose a relevant exposure and subsequently a potential health risk. The aim of this study was to determine the relative bioavailability of certain phthalates, such as di(2-ethylhexyl) phthalate (DEHP), di-isononyl phthalate (DINP) and the non-phthalate plasticizer diisononyl 1,2-cyclohexanedicarboxylic acid (DINCH®, Hexamoll®), after ingestion of dust. Seven 5-week-old male piglets were fed five different dust samples collected from daycare centers. Overall, 0.43 g to 0.83 g of dust sieved to 63 µm were administered orally. The piglets' urine was collected over a period of 38 h. The excreted metabolites were quantified using an LC-MS/MS method. The mean uptake rates of the applied doses for DEHP, DINP, and DINCH® were 43% ± 11%, 47% ± 26%, and 9% ± 3.5%, respectively. The metabolites of DEHP and DINP showed maximum concentrations in urine after three to five hours, whereas the metabolites of DINCH®, reached maximum concentrations 24 h post-dose. The oral bioavailability of the investigated plasticizers was higher compared to the bioaccessibility reported from in vitro digestion tests. Furthermore, the bioavailability of DEHP did not vary substantially between the dust samples, whereas a dose-dependent saturation process for DINP was observed. In addition to other intake pathways, dust could be a source of plasticizers in children using the recent intake rates for dust ingestion.

Urinary metabolites of phthalates and di-iso-nonyl cyclohexane-1,2-dicarboxylate (DINCH)-Czech mothers' and newborns' exposure biomarkers.

To assess human exposure to hazardous diesters of phthalic acid and their substitute di-iso-nonyl cyclohexane-1,2-dicarboxylate (DINCH), concentrations of their metabolites in urine should be determined. For the purpose of this biomonitoring study, a quick and easy sample preparation procedure for the simultaneous determination of eight phthalate and four DINCH metabolites in urine has been implemented and validated. Following the enzymatic hydrolysis and dilution with methanol, the sample is ready for the analysis by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The limits of quantification of this method ranged from 0.15 to 0.4 ng/mL urine with recoveries of 60-126% and repeatability in the range of 1-11%. The validated method was subsequently used for the analysis of urine samples collected from mothers and their newborn children living in two localities of the Czech Republic (Karvina and Ceske Budejovice, 2013-2014). Median concentrations of all measured metabolites (∑metabolites) were slightly lower in the urine samples collected from children (77.7 ng/mL urine) compared to their mothers (115.3 ng/mL urine), but no correlation was found between the concentrations of target compounds in children's and mothers' urine samples. The analyte with the highest concentration was monobutyl phthalate (MBP), with the median concentration of 32.1 ng/mL urine in the urine samples collected from mothers and 17.2 ng/mL urine in the samples collected from their children. This compound was also found in almost all of the measured samples. On the other hand, mono-isononyl-cyclohexane-1,2-dicarboxylate (MINCH) was not found in any urine sample. The most contaminated samples were collected from children living in the Karvina locality (median ∑metabolites 103.2 ng/mL urine), where the mono (2-ethyl-5-carboxypentyl) phthalate (cx-MEHP) compound contributed 43% to the total content of phthalate metabolites in newborns' urine. The results from our study are comparable with concentrations of the target compounds from Norway and Germany and lower compared to the results concluded in Sweden.

Development and validation of a bioanalytical assay based on liquid chromatography-tandem mass spectrometry for measuring biomarkers of exposure of alternative plasticizers in human urine and serum.

Alternative plasticizers (APs) have been increasingly used in the last decade to replace conventional phthalate esters, in particular di(2-ethylhexyl) phthalate (DEHP), due to the toxicity of the latter. However, there is currently very little data about the toxicity of and exposure to APs. No method exists so far for the analysis of multiple exposure biomarkers. The objective of this work consisted in developing a simple bioanalytical procedure for the analysis of multiple exposure biomarkers of APs in human urine and serum. Focus was set on metabolites of di(2-propylheptyl) phthalate (DPrHpP), di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH), di(2-ethylhexyl) terephthalate (DEHTP) and di-2-ethylhexyl adipate (DEHA). A sample preparation protocol was developed and optimized using Oasis HLB solid-phase extraction (SPE) cartridges. Subsequently, an instrumental method based on liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) was optimized. Following established guidelines, the sample preparation and instrumental methods were validated in terms of recovery, matrix effects, carry-over, linearity, limits of quantification, within- and between-run precision and trueness. Obtained results were satisfactory for all compounds except for one of the metabolites of DEHA (i.e., mono(2-ethylhexyl) adipate (MEHA)). A pilot biomonitoring study was carried out to assess the method's ability to detect and quantify target analytes in human urine and serum. In urine, most analytes could be detected with frequencies ranging from 8% for mono(2-ethyl-5-hydroxyhexyl) adipate (OH-MEHA) and cyclohexane-1,2-dicarboxylic mono hydroxyisononyl ester (OH-MINCH) to 92% for mono(2-ethyl-5-oxohexyl) adipate (oxo-MEHA), whilst most compounds could not be detected in serum, except for mono(2-ethylhexyl) terephthalate (MEHTP) and mono-(2-propyl-6-hydroxyheptyl) phthalate (OH-MPrHpP) which were detected in all samples. The obtained results show that the developed method can be used to simultaneously analyse multiple exposure biomarkers to APs in human urine and serum.

Urinary biomarker panel for diagnosing patients with depression and anxiety disorders.

Available data indicate that patients with depression and anxiety disorders are likely to be at greater risk for suicide. Therefore, it is important to correctly diagnose patients with depression and anxiety disorders. However, there are still no empirical laboratory methods to objectively diagnose these patients. In this study, the multiple metabolomics platforms were used to profile the urine samples from 32 healthy controls and 32 patients with depression and anxiety disorders for identifying differential metabolites and potential biomarkers. Then, 16 healthy controls and 16 patients with depression and anxiety disorders were used to independently validate the diagnostic performance of the identified biomarkers. Finally, a panel consisting of four biomarkers-N-methylnicotinamide, aminomalonic acid, azelaic acid and hippuric acid-was identified. This panel was capable of distinguishing patients with depression and anxiety disorders from healthy controls with an area under the receiver operating characteristic curve of 0.977 in the training set and 0.934 in the testing set. Meanwhile, we found that these identified differential metabolites were mainly involved in three metabolic pathways and five molecular and cellular functions. Our results could lay the groundwork for future developing a urine-based diagnostic method for patients with depression and anxiety disorders.

The urinary metabolites of DINCH® have an impact on the activities of the human nuclear receptors ERα, ERß, AR, PPARα and PPARγ.

DINCH® (di-isononyl cyclohexane-1,2-dicarboxylate) is a non-phthalate plasticizer that has been developed to replace phthalate plasticizers such as DEHP (di-2-ethylhexyl phthalate) or DINP (di-isononyl phthalate). DINCH® is metabolized to its corresponding monoester and subsequently to oxidized monoester derivatives. These are conjugated to glucuronic acid and subject to urinary excretion. In contrast to DINCH®, there are almost no toxicological data available regarding its primary and secondary metabolites. The present study aimed at the characterization of potential endocrine properties of DINCH® and five DINCH® metabolites by using reporter gene assays to monitor the activity of the human nuclear receptors ERα, ERß, AR, PPARα and PPARγ in vitro. DINCH® itself did not have any effect on the activity of these receptors whereas DINCH® metabolites were shown to activate all these receptors. In the case of AR, DINCH® metabolites predominantly enhanced dihydrotestosterone-stimulated AR activity. In the H295R steroidogenesis assay, neither DINCH® nor any of its metabolites affected estradiol or testosterone synthesis. In conclusion, primary and secondary DINCH® metabolites exert different effects at the molecular level compared to DINCH® itself. All these in vitro effects of DINCH® metabolites, however, were only observed at high concentrations such as 10 µM or above which is about three orders of magnitude above reported DINCH® metabolite concentrations in human urine. Thus, the in vitro data do not support the notion that DINCH® or any of the investigated metabolites may exert considerable endocrine effects in vivo at relevant human exposure levels.

High-Throughput Metabolomics for Discovering Potential Metabolite Biomarkers and Metabolic Mechanism from the APPswe/PS1dE9 Transgenic Model of Alzheimer's Disease.

Alzheimer's disease (AD), a neurodegenerative disorder, is the major form of dementia. As AD is an irreversible disease, it is necessary to focus on earlier intervention. However, the potential biomarkers of preclinical AD are still not clear. In this study, urinary metabolomics based on ultra-high-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry was performed for delineating the metabolic changes and potential early biomarkers in APPswe/PS1dE9 (APP/PS1) transgenic mice. A total of 24 differentially regulated metabolites were identified when comparing transgenic mice to wild-type mice using multivariate statistical analysis. Among them, 10 metabolites were significantly upregulated and 14 metabolites were downregulated. On the basis of these potential biomarkers, metabolic pathway analysis found that pentose and glucuronate interconversions, glyoxylate and dicarboxylate metabolism, starch and sucrose metabolism, the citrate cycle, tryptophan metabolism, and arginine and proline metabolism were disturbed in APP/PS1 mice. Our study revealed that levels of endogenous metabolites in the urine of APP/PS1 mice changed prior to the emergence of learning and cognitive impairment, which may be associated with abnormal nitric oxide production pathways and metabolic disorders of monoaminergic neurotransmitters. In conclusion, this study showed that metabolomics provides an early indicator of disease occurrence for AD.

Exposure of Portuguese children to the novel non-phthalate plasticizer di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate (DINCH).

Di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate (DINCH) is used as substitute for high molecular weight phthalate plasticizers such as di-(2-ethylhexyl) phthalate (DEHP) and di-(iso-nonyl) phthalate (DINP). Due to a rapid substitution process we have to assume omnipresent and increasing DINCH exposures. The aim of this study was to evaluate DINCH exposure in 112 children (4-18years old) from Portugal, divided in two groups: 1) normal-/underweight following the usual diet; and 2) obese/overweight but under strict nutritional guidance. First morning urine samples were collected during the years 2014 and 2015. Oxidized DINCH metabolites (OH-MINCH, oxo-MINCH, cx-MINCH) were analyzed after enzymatic hydrolysis via on-line HPLC-MS/MS with isotope dilution quantification. We detected DINCH metabolites in all analyzed samples. Urinary median (95th percentile) concentrations were 2.14µg/L (15.91) for OH-MINCH, followed by 1.10µg/L (7.54) for oxo-MINCH and 1.08µg/L (7.33) for cx-MINCH. We observed no significant differences between the two child-groups; only after creatinine adjustment, we found higher metabolite concentrations in the younger compared to the older children. Median (95th percentile) daily DINCH intakes were in the range of 0.37 to 0.76 (2.52 to 5.61) µg/kg body weight/day depending on calculation model and subpopulation. Body weight related daily intakes were somewhat higher in Group 1 compared to Group 2, irrespective of the calculation model. However, in terms of absolute amounts (µg/day), DINCH intakes were higher in Group 2 compared to Group 1. In regard to age, we calculated higher intakes for the younger children compared to older children, but only with the creatinine-based model. This new data for southern European, Portuguese children adds information to the scarce knowledge on DINCH, confirming omnipresent exposure and suggesting higher exposures in children than adults. Significant sources and routes of exposure have yet to be unveiled. For now, all calculated daily intakes are far below established health benchmark levels (TDI, RfD). However, rapidly increasing exposures have to be expected over the next years.

Additional oxidized and alkyl chain breakdown metabolites of the plasticizer DINCH in urine after oral dosage to human volunteers.

Hexamoll® DINCH® (diisononyl-cyclohexane-1,2-dicarboxylate) is a new high molecular weight plasticizer and a non-aromatic phthalate substitute. In this follow-up study, we further investigated the extensive oxidative metabolism of Hexamoll® DINCH® after oral dosage of 50 mg to three male volunteers (0.552-0.606 mg/kg body weight). Urine samples were consecutively collected over 48 h post-dose. Chemical analysis was carried out by HPLC-MS/MS with labeled internal standards. New metabolites were tentatively identified and quantified via fragmentation analogies and new standard substances. In addition to the five urinary DINCH metabolites previously reported by us, we identified two groups of extensively oxidized metabolites characterized (a) by multiple side chain oxidation and breakdown and (b) by hydroxylation at the cyclohexane ring. The five newly identified carboxylated breakdown metabolites represented in sum 5.12 ± 0.49 % of the applied dose. MCHxCH (cyclohexane-1,2-dicarboxylic acid mono carboxyhexyl ester) was identified as a major metabolite (2.71 ± 0.34 %) and thus represents the second most important specific metabolite of DINCH after OH-MINCH (10.7 ± 2.1 %). Less than 1 % was excreted as ring-hydroxylated metabolites (four metabolites identified). Based upon a new reference standard, we can also update oxo-MINCH to 2.6 % of the applied dose. This follow-up study increases the total amount of the recovered dose from 39.2 to 45.7 % and describes a new major metabolite (MCHxCH) of DINCH that can be used as an additional valuable and specific biomarker to assess DINCH® exposure in future human biomonitoring studies.

FODMAPs alter symptoms and the metabolome of patients with IBS: a randomised controlled trial.

OBJECTIVE: To gain mechanistic insights, we compared effects of low fermentable oligosaccharides, disaccharides and monosaccharides and polyols (FODMAP) and high FODMAP diets on symptoms, the metabolome and the microbiome of patients with IBS. DESIGN: We performed a controlled, single blind study of patients with IBS (Rome III criteria) randomised to a low (n=20) or high (n=20) FODMAP diet for 3 weeks. Symptoms were assessed using the IBS symptom severity scoring (IBS-SSS). The metabolome was evaluated using the lactulose breath test (LBT) and metabolic profiling in urine using mass spectrometry. Stool microbiota composition was analysed by 16S rRNA gene profiling. RESULTS: Thirty-seven patients (19 low FODMAP; 18 high FODMAP) completed the 3-week diet. The IBS-SSS was reduced in the low FODMAP diet group (p<0.001) but not the high FODMAP group. LBTs showed a minor decrease in H2 production in the low FODMAP compared with the high FODMAP group. Metabolic profiling of urine showed groups of patients with IBS differed significantly after the diet (p<0.01), with three metabolites (histamine, p-hydroxybenzoic acid, azelaic acid) being primarily responsible for discrimination between the two groups. Histamine, a measure of immune activation, was reduced eightfold in the low FODMAP group (p<0.05). Low FODMAP diet increased Actinobacteria richness and diversity, and high FODMAP diet decreased the relative abundance of bacteria involved in gas consumption. CONCLUSIONS: IBS symptoms are linked to FODMAP content and associated with alterations in the metabolome. In subsets of patients, FODMAPs modulate histamine levels and the microbiota, both of which could alter symptoms. TRIAL REGISTRATION NUMBER: NCT01829932.

Evaluation of exposure to phthalate esters and DINCH in urine and nails from a Norwegian study population.

Phthalate esters (PEs) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) used as additives in numerous consumer products are continuously released into the environment, leading to subsequent human exposure which might cause adverse health effects. The human biomonitoring approach allows the detection of PEs and DINCH in specific populations, by taking into account all possible routes of exposure (e.g. inhalation, transdermal and oral) and all relevant sources (e.g. air, dust, personal care products, diet). We have investigated the presence of nine PE and two DINCH metabolites and their exposure determinants in 61 adult residents of the Oslo area (Norway). Three urine spots and fingernails were collected from each participant according to established sampling protocols. Metabolite analysis was performed by LC-MS/MS. Metabolite levels in urine were used to back-calculate the total exposure to their corresponding parent compound. The primary monoesters, such as monomethyl phthalate (MMP, geometric mean 89.7ng/g), monoethyl phthalate (MEP, 104.8ng/g) and mono-n-butyl phthalate (MnBP, 89.3ng/g) were observed in higher levels in nails, whereas the secondary bis(2-ethylhexyl) phthalate (DEHP) and DINCH oxidative metabolites were more abundant in urine (detection frequency 84-100%). The estimated daily intakes of PEs and DINCH for this Norwegian population did not exceed the established tolerable daily intake and reference doses, and the cumulative risk assessment for combined exposure to plasticizers with similar toxic endpoints indicated no health concerns for the selected population. We found a moderate positive correlation between MEP levels in 3 urine spots and nails (range: 0.56-0.68). Higher frequency of personal care products use was associated with greater MEP concentrations in both urine and nail samples. Increased age, smoking, wearing plastic gloves during house cleaning, consuming food with plastic packaging and eating with hands were associated with higher levels in urine and nails for some of the metabolites. In contrast, frequent hair and hand washing was associated with lower urinary levels of monoisobutyl phthalate (MiBP) and mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), respectively.

Relationship between adipic acid concentration and the core symptoms of autism spectrum disorders.

Dicarboxylic acids are an important source of information about metabolism and potential physiopathological alterations in children with autism spectrum disorders (ASDs). We measured the concentration between dicarboxylic adipic and suberic acids in children with an ASD and typically-developing (TD) children and analyzed any relationships between the severity of the core symptoms of ASDs and other clinical features (drugs, supplements, drugs, or diet). The core symptoms of autism were evaluated using the DSM-IV criteria, and adipic acid and suberic acid were measured in urine samples. Overall, no increase in the concentration of adipic acid in children with ASDs compared to TD children, however when considering vitamin B supplementation in ASD there were significantly increased level of urinary adipic acid in children with an ASD not taking vitamin B supplementation compared to supplemented children or to TD children. No significant difference were observed in suberic acid. Interestingly, the increase in adipic acid concentration was significantly and indirectly correlated with the severity of the deficit in socialization and communication skills in children with an ASD. Therefore, therapeutic treatments aimed at decreasing adipic acid concentration might not be beneficial for treating the core symptoms of ASDs.

Urinary toxicokinetics of di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH(®)) in humans following single oral administration.

Phthalates such as di-2-ethylhexyl phthalate (DEHP) were restricted due to their toxic mainly reprotoxic effects. Therefore compounds such as di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH(®)) substitute these phthalates and the exposure of humanes to substitutes may occur. Here, kinetic data are presented to assess the exposure of humans. Male and female volunteers excreted nearly the complete orally administered dose (1mg/kg b.w. corresponding to the tolerable daily intake of EFSA) of di-(isononyl)-cyclohexane-1,2-dicarboxylate within 70 h. More than 75% were excreted within 24h. Besides the main metabolite cyclohexane-1,2-dicarboxylic acid (CHDA) quantitated after hydrolysis four further metabolites of DINCH(®) are determined. Cyclohexane-1,2-dicarboxylic acid-mono-(7-hydroxy-4-methyl)octyl ester (OH-MINCH) is the main secondary metabolite with about 14% of the administered dose. Differences in excretion of all metabolites between male and females are small. Based on the generated toxicokinetic data exposure of 20 humans is recalculated from their spot urine sample collected in 2014 and the exposure are clearly below the current tolerable daily intake of 1mg/kg b.w.