An Untargeted Urine Metabolomics Approach for Autologous Blood Transfusion Detection.

PURPOSE: Autologous blood transfusion is performance enhancing and prohibited in sport but remains difficult to detect. This study explored the hypothesis that an untargeted urine metabolomics analysis can reveal one or more novel metabolites with high sensitivity and specificity for detection of autologous blood transfusion. METHODS: In a randomized, double-blinded, placebo-controlled, crossover design, exercise-trained men (n = 12) donated 900 mL blood or were sham phlebotomized. After 4 wk, red blood cells or saline were reinfused. Urine samples were collected before phlebotomy and 2 h and 1, 2, 3, 5, and 10 d after reinfusion and analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. Models of unique metabolites reflecting autologous blood transfusion were attained by partial least-squares discriminant analysis. RESULTS: The strongest model was obtained 2 h after reinfusion with a misclassification error of 6.3% and 98.8% specificity. However, combining only a few of the strongest metabolites selected by this model provided a sensitivity of 100% at days 1 and 2 and 66% at day 3 with 100% specificity. Metabolite identification revealed the presence of secondary di-2-ethylhexyl phtalate metabolites and putatively identified the presence of (iso)caproic acid glucuronide as the strongest candidate biomarker. CONCLUSIONS: Untargeted urine metabolomics revealed several plasticizers as the strongest metabolic pattern for detection of autologous blood transfusion for up to 3 d. Importantly, no other metabolites in urine seem of value for antidoping purposes.

5-Hydroxyhexanoic Acid Predicts Early Renal Functional Decline in Type 2 Diabetes Patients with Microalbuminuria.

BACKGROUND/AIMS: Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. Microalbuminuria (MA) is widely used to predict early progressive renal function decline (ERFD) of DN in type 2 diabetes mellitus (T2D) patients, but the sensitivity and specificity of MA have been questioned. Here, we determined the urine metabolites differences between T2D patients with MA who maintained stable renal function and those who progressed to ERFD in order to identify specific biomarkers of the progression of renal dysfunction. METHODS: A total of 102 T2D patients with MA and normal renal function at baseline were followed up for 5-6 years. Of these, 52 patients were selected and classified into two groups according to the later renal function; 25 patients who experienced ERFD were regarded as the progressive group, while 27 patients who maintained stable renal function were considered as the stable group. In the pilot study, untargeted, broad-spectrum urine metabolomics was performed on the urine of 12 subjects from the progressive group (5 patients as "progressors") and stable group (7 patients as "non-progressors") to discover candidate markers. We then used a targeted metabolomics analysis to identify the selected markers in the urine of an additional 40 patients (20 from the progressive group as cases, and 20 from the stable group as controls) in the validation study. RESULTS: A total of 318 known metabolites were detected in the pilot study and 6 metabolites with significant difference between progressors and non-progressors were identified. The levels of 4 metabolites, including azelaic acid, adipic acid, 5-hydroxyhexanoic acid, and L-tryptophan decreased significantly, while levels of L-pyroglutamic acid and D-norvaline increased observably in the progressors compared with non-progressors. Furthermore, in the validation study, 6 metabolites were confirmed by quantitative measurements and their concentrations were consistent with the changes in the pilot study. Concentrations of L-pyroglutamic acid and D-norvaline still increased in the cases, but were not statistically significant. Of the 4 metabolites with decreased concentrations among the cases, only 5-hydroxyhexanoic acid remained statistically significant while the other 3 metabolites did not differ between cases and controls. CONCLUSION: We have identified urine metabolites and shown that 5-hydroxyhexanoic acid can be used as a predictor of progression of ERFD in T2D patients with MA. This finding provides the new perspective that 5-hydroxyhexanoic acid may be useful to identify T2D patients with MA who are at risk of ERFD.

Perfluoroalkyl acids in paired serum, urine, and hair samples: Correlations with demographic factors and dietary habits.

We analyzed paired serum, urine, and hair samples from 94 Korean children and adults to investigate levels of 11 perfluoroalkyl acids (PFAAs). The effects of demographic factors and dietary habits on PFAA exposure were also assessed based on the paired samples. The total PFAA concentrations were 2.4-31 ng/mL in serum, not detected-9.5 ng/mL in urine, and 0.48-15 ng/g in hair. Levels of perfluoropentanoic acid (PFPeA) and perfluorohexanoic acid (PFHxA), which have short carbon chains, were 1.5-5 fold higher in urine and hair than in serum. The PFAA concentrations in serum exhibited a decreasing trend with age from young childhood to adolescence, followed by an increasing trend after adolescence. For most PFAA species, concentrations in serum were higher in adult males than in adult females (p < 0.01). No sex difference was evident in the urine and hair samples. In addition, there was no age difference in the urine samples, but in the hair samples, we observed higher concentrations of PFAAs in children than in the other age groups (p < 0.01). The consumption rates of fish and water showed significant correlations with serum (positive correlation) and hair (negative) concentrations, respectively. No relationships between serum and hair/urine levels for most PFAAs were observed, except between serum and hair levels for perfluorooctanoic acid (PFOA).

Association between metabolic profiles in urine and bone mineral density of pre- and postmenopausal Chinese women.

OBJECTIVE: In the present study, we aimed to characterize the pathological development of menopausal osteoporosis, as well as to explore potential biomarkers and metabolic pathways involved in osteoporosis. METHODS: Urine samples from 322 female participants categorized by menopause status and different bone conditions were collected and analyzed based on a gas chromatography-mass spectrometry (GC-MS) approach. Multivariate and univariate statistical analyses were carried out for urinary metabolomic profile characterization and comparison. RESULTS: Seventeen metabolites in the low bone mineral density (BMD) groups were clearly differentiated from those in normal BMD groups. Among these 17 differentiating metabolites, taurine, ß-alanine, and 5-hydroxycaproic acid were found to be potential biomarkers of osteoporosis. The taurine metabolic pathway and the ß-alanine metabolic pathway were found to be related to menopause and bone loss. CONCLUSIONS: Based on the GC-MS metabolomic platform, four typical pathological phases during the progression of postmenopausal osteoporosis were described. Several differentiating metabolites and metabolic pathways were found to be closely related to the pathology of postmenopausal osteoporosis. Our results provided a solid foundation for further studies on early diagnosis and pathomechanistic evaluation.

Fast and simple screening for the simultaneous analysis of seven metabolites derived from five volatile organic compounds in human urine using on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry.

Recently, the International Agency for Research on cancer classified outdoor air pollution and particulate matter from outdoor air pollution as carcinogenic to humans (IARC Group 1), based on sufficient evidence of carcinogenicity in humans and experimental animals and strong mechanistic evidence. In particular, a wide variety of volatile organic compounds (VOCs) are volatized or released into the atmosphere and can become ubiquitous, as they originate from many different natural and anthropogenic sources, such as paints, pesticides, vehicle exhausts, cooking fumes, and tobacco smoke. Humans may be exposed to VOCs through inhalation, ingestion, or dermal contact, which may increase the risk of leukemia, birth defects, neurocognitive impairment, and cancer. Therefore, the focus of this study was the development of a simple, effective and rapid sample preparation method for the simultaneous determination of seven metabolites (6 mercaptic acids+t,t-muconic acid) derived from five VOCs (acrylamide, 1,3-butadiene, acrylonitrile, benzene, and xylene) in human urine by using automated on-line solid-phase extraction (SPE) coupled with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). An aliquot of each diluted urinary sample was directly injected into an autosampler through a trap column to reduce contamination, and then the retained target compounds were eluted by back-flush mode into an analytical column for separation. Negative electrospray ionization tandem mass spectrometry was utilized for quantification. The coefficients of correlation (r(2)) for the calibration curves were greater than 0.995. Reproducibility was assessed by the precision and accuracy of intra-day and inter-day precision, which showed results for coefficient of variation (CV) that were low 0.9 to 6.6% and 3.7 to 8.5%, respectively, and results for recovery that ranged from 90.8 to 108.9% and 92.1 to 107.7%, respectively. The limits of detection (LOD) and limits of quantification (LOQ) were determined to within 0.010 to 0.769 ng mL(-1) and 0.033 to 2.564 ng mL(-1) in this study. A stability study test included 3 freeze/thaw cycles during short-term storage at room temperature for 36 h and long-term storage at -20 °C for 1 month, and the CV (coefficient of variation) showed less than 8.4, 7.4 and 9.7%, respectively. To the best of our knowledge, this is the first study to provide simple, small injection volumes (40 µL) and a rapid LC-MS/MS method combined with an on-line SPE step for the simultaneous detection, identification, and quantification of seven metabolites derived from five VOCs in human urine for evaluation of the future risk of human exposure to volatile organic compounds.

A Novel Urinary Metabolite Signature for Non-invasive Post-stroke Depression Diagnosis.

Post-stroke depression (PSD) is the most common psychiatric complication in stroke survivors that has been associated with increased physical disability, distress, poor rehabilitation, and suicidal ideation. However, there are still no biomarkers available to support objective laboratory testing for this disorder. Here, a GC-MS-based urinary metabolomics approach was used to characterize the urinary metabolic profiling of PSD (stroke) subjects and non-PSD (health controls) subjects in order to identify and validate urinary metabolite biomarkers for PSD. Six metabolites, azelaic acid, glyceric acid, pseudouridine, 5-hydroxyhexanoic acid, tyrosine, and phenylalanine, were defined as biomarkers. A combined panel of these six urinary metabolites could effectively discriminate between PSD subjects and non-PSD subjects, achieving an area under the receiver-operating characteristic curve (AUC) of 0.961 in a training set (n = 72 PSD subjects and n = 146 non-PSD subjects). Moreover, this urinary biomarker panel was capable of discriminating blinded test samples (n = 58 PSD patients and n = 109 non-PSD subjects) with an AUC of 0.954. These findings suggest that a urine-based laboratory test using these biomarkers may be useful in the diagnosis of PSD.

Toxicokinetics of ammonium perfluorohexanoate.

Excretion patterns and rates of ammonium perfluorohexanoate (APFHx) after administration of a single and multiple (14 days) oral dose(s) at 50 mg/kg to male and female mice and rats were examined. The test substance was [(14)C]-labeled APFHx. After a single oral administration, total excretion was rapid, with mean recoveries of over 90% of the dose at 24 hours after administration, irrespective of gender or species. The major route of elimination was via the urine (means of percentage recovery between 73.0 and 90.2% of the dose), followed by the feces (means of percentage recovery between 7.0 and 15.5% of the dose). Elimination via expired air was negligible. For the multiple dose tests, multiple (13 daily doses) oral administration of APFHx was followed by a single oral administration of [(14)C]-APFHx. Excretion was rapid, with mean recoveries of over 90% of the administered dose (mean values >95% of the ultimately recovered material) at 24 hours after dosing, irrespective of gender or species. The major route of elimination was via the urine (means of percentage recovery between 77.8 and 83.4% of the dose), followed by the feces (means of percentage recovery between 9.6 and 12.9% of the dose).

Absorption, distribution, metabolism, and excretion of [1-¹4C]-perfluorohexanoate ([¹4C]-PFHx) in rats and mice.

The absorption, tissue distribution, elimination, and metabolism of [1-¹4C]-PFHx in rats and mice dosed orally at 2 or 100 mg/kg was evaluated following a single dose or after 14 consecutive doses. Absorption was rapid in rats as evidenced by a short time to maximum concentration (C(max)) of 30 min in male rats and 15 min in female rats at both the 2 and 100mg/kg dose level. The plasma elimination half-life was somewhat longer in males (1.5-1.7 h) than in females (0.5-0.7 h). Absorption in the mouse was also rapid with the maximum plasma concentration occurring between 15 and 30 min after dosing. The maximum concentration was not appreciably different between male and female mice (8 µg equiv./g at 2 mg/kg; ~350 µg equiv./g at 100 mg/kg). The primary route of elimination was via the urine. PFHx was not metabolized in rat or mouse hepatocytes, nor were any metabolites observed after oral dosing in either rodent species. Essentially 100% of the dose was eliminated in urine within 24 h demonstrating that PFHx is readily absorbed and bioavailability approaches 100%, even at a dose as high as 100 mg/kg. The route and extent of elimination was unchanged after 14 days of daily dosing. Tissues were collected at three time points (rat: 0.5, 2, and 24 h; mice: 0.25, 1, and 24 h) after dosing to investigate the tissue clearance kinetics of PFHx following a single dose at 2 or 100 mg/kg. In all tissues except skin, PFHx was not quantifiable 24 h after dosing in both sexes of the two species.

Biological monitoring of exposure to di(2-ethylhexyl) phthalate in six French factories: a field study.

OBJECTIVE: The aim of this study was to assess, by biological monitoring, workers' exposure to di(2-ethylhexyl) phthalate (DEHP) in the flexible-PVC industry in France to provide additional occupational exposure data, which are particularly scarce. METHOD: Over 5 days of pre-and post-shift sampling, three urinary metabolites of DEHP, mono (2-ethylhexyl) phthalate (MEHP), mono (5-carboxy-2-ethylpentyl) phthalate (5cx-MEPP) and 2-ethylhexanoic acid (2-EHA) were quantified in 62 workers and 29 controls from six factories. Analyses were performed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) after on-line extraction. RESULTS: Median concentrations of the pre- and post-shift urinary samples in the exposed workers were 12.6 and 28.7 µg/l for MEHP, 38.6 and 84.4 µg/l for 5cx-MEPP and 20.4 and 70.6 µg/l for 2-EHA, respectively. In the controls, the corresponding values were 4.8 and 4.7 µg/l for MEHP, 15.1 and 12.4 µg/l for 5cx-MEPP and 21.8 and 20.5 µg/l for 2-EHA, respectively. There was a significant increase (Mann-Whitney U-test, P < 0.05) of post-shift excretion in the exposed workers versus the unexposed controls and in the post-shift versus pre-shift concentrations only in the exposed workers. Values of 250 and 500 µg/l (100 and 280 µ/g creatinine) for MEHP and 5cx-MEPP, respectively, are proposed as guidance values. CONCLUSION: There is clear evidence of occupational exposure of workers in these factories. The guideline values proposed should prevent high exposures in the soft PVC industry, particularly in factories where DEHP compounds or plastisols are employed. An epidemiological survey is needed to complete the DEHP risk assessment.

Visual evoked potentials in succinate semialdehyde dehydrogenase (SSADH) deficiency.

In mammals, increased GABA in the central nervous system has been associated with abnormalities of visual evoked potentials (VEPs), predominantly manifested as increased latency of the major positive component P100. Accordingly, we hypothesized that patients with a defect in GABA metabolism, succinate semialdehyde dehydrogenase (SSADH) deficiency (in whom supraphysiological levels of GABA accumulate), would manifest VEP anomalies. We evaluated VEPs on two patients with confirmed SSADH deficiency. Whereas the P100 latencies and amplitudes for binocular VEP analyses were within normal ranges for both patients, the P100 latencies were markedly delayed for left eye (OS) (and right eye (OD), patient 1) and monocular OS (patient 2): 134-147 ms; normal <118 ms. We hypothesize that elevated GABA in ocular tissue of SSADH patients leads to a use-dependent downregulation of the major GABAergic receptor in eye, GABA(C), and that the VEP recordings' abnormalities, as evidenced by P100 latency and/or amplitude measurements, may be reflective of abnormalities within visual systems. This is a preliminary finding that may suggest the utility of performing VEP analysis in a larger sample of SSADH-deficient patients, and encourage a neurophysiological assessment of GABA(C) receptor function in Aldh5a1(-/-) mice to reveal new pathophysiological mechanisms of this rare disorder of GABA degradation.

Biological monitoring of occupational exposure to di(2-ethylhexyl) phthalate: survey of workers exposed to plastisols.

OBJECTIVE: The aim of this study was to perform a biological monitoring survey of workers exposed to di(2-ethylhexyl)phthalate (DEHP) in a factory using polyvinyl chloride plastisols and to contribute additional occupational data of exposure particularly sparse in the industrial sectors where this plasticizer is used. METHOD: Three urinary metabolites of DEHP, mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate (MCEPP) and 2-ethylhexanoic acid (2-EHA) were quantified in five workers using a plastisol (containing 33% of DEHP) and in five unexposed workers (controls), during 5 days with pre- and post-shift sampling. Analyses were performed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) with on-line extraction. RESULTS: Median concentrations of pre- and post-shift urinary samples in the exposed workers (n = 25) were 16.1 and 55.9 microg/l for MEHP, 37.6 and 103.7 microg/l for MCEPP and 46.3 and 72.1 microg/l for 2-EHA, respectively. In the controls (n = 19), the corresponding values were 12.0 and 10.4 microg/l for MEHP, 38.1 and 11.4 microg/l for MCEPP and 31.9 and 46.0 microg/l for 2-EHA, respectively. There is a significant increase (Mann-Whitney U-test, P < 0.05) of post-shift excretion in the exposed workers versus unexposed controls and in post-shift versus pre-shift concentrations only in the exposed workers. CONCLUSION: MEHP and MCEPP are shown to be suitable biomarkers to assess DEHP exposure while 2-EHA, less specific but classified in the category 3 of the European Union (EU) reproductive toxicants, is also an interesting biomarker. There is clear evidence of occupational exposure of workers in this factory.

Urinary 2-ethyl-3-oxohexanoic acid as major metabolite of orally administered 2-ethylhexanoic acid in human.

Human metabolism of 2-ethylhexanoic acid (2-EHA), which is a known metabolite of important phthalates, was investigated using 2-EHA-contaminated food. The results of our studies reveal that the major catabolic pathway of 2-EHA in human is beta-oxidation. The dominant final urinary metabolite was identified and quantified as 3-oxo-2-ethylhexanoic acid (3-oxo-2-EHA), but only after immediate methylation of the extract from urine and prior to GC-MS analysis. Former studies without the precaution of immediate methylation had found 4-heptanone as the major metabolite, which is obviously an artifact arising from the decarboxylation of 3-oxo-2-EHA.

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC-MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1,000 microg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1 +/- 13.5 microg/l for 2-ethylhexanoic acid, 104.8 +/- 80.6 microg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2 +/- 389.5 microg/l for 2-ethyl-3-oxohexanoic acid.

Metabolism of 2-ethylhexanoic acid administered orally or dermally to the female Fischer 344 rat.

1. Excretion balance studies were conducted with 2-ethylhexanoic acid (EHA) in the female Fischer 344 rat following single high (1 g/kg) or low (0.1 g/kg) oral doses of [2-14C-hexyl]EHA, following repeated oral dosing with unlabelled EHA and a final [14C]EHA oral dose at the low dose level, following dermal exposure with a high (1 g/kg) and low (0.1 g/kg) applied dose of [14C]EHA, and following a 1 mg/kg i.v. dose of [14C]EHA. 2. Oral, i.v. and dermal doses were eliminated rapidly, predominantly in the urine during the first 24 h following dosing. 3. After oral dosing of 0.1 g/kg, the mean peak blood level was 85.1 micrograms equivalents EHA/g. Maximum blood concentrations were detected at either 15 or 30 min in individual animals. After dermal application of 0.1 g/kg, the mean peak blood level of 7.9 micrograms equivalents EHA/g was attained at 8 h. 4. Occlusive dermal exposure caused damage to the epidermis in the first 24 h after application and resulted in dermal absorption of 70% relative to i.v. dosing, based on the ratio of percent dose in excreta. 5. Dermal application followed by prompt washing of the skin resulted in recovery of 101.9% from the skin surface and < 0.2% in the excreta. 6. The major urinary metabolites were the glucuronide of EHA, 2-ethyl-1,6-hexanedioic acid (namely 2-ethyladipic acid), 2-ethyl-5-hydroxyhexanoic acid, 2-ethyl-6-hydroxyhexanoic acid and ethylketohexanoic acid. Evidence for metabolism via beta-oxidation was also found, consistent with the incorporation of EHA into normal cellular intermediary metabolism.

CYP enzymes catalyze the formation of a terminal olefin from 2-ethylhexanoic acid in rat and human liver.

1. The metabolism of 2-ethylhexanoic acid (2-EHA) was studied in rat, mouse and human liver microsomes in vitro. The metabolites of 2-EHA were identified as methylated derivatives by gas chromatography-mass spectrometry. 2. 2-Ethyl-1,6-hexanedioic acid was the main metabolite produced in rat, mouse and human liver microsomes. Unsaturated 2-ethyl-5-hexenoic acid, a terminal olefin, was produced only in human liver microsomes and phenobarbital-induced rat liver microsomes. The cytochrome P450 (CYP) inhibitors metyrapone, SKF 525A, triacetyloleandomycin (TAO), quinidine and the cytochrome P450 reductase antibody abolished its formation both in rat and human microsomes. 3. The metabolites were analyzed also in vivo in urine of 2-EHA-exposed rats and in urine of sawmill workers exposed occupationally to 2-EHA. Both rat and human urine contained 2-ethyl-1,6-hexanedioic acid as the main metabolite and also 2-ethyl-5-hexenoic acid. Metyrapone, SKF 525A and TAO all decreased drastically the formation of 2-ethyl-5-hexenoic acid in the rat. 4. The data indicate that (1) several CYP families (CYP2A, CYP2B, CYP2D and CYP3A) could be responsible for the hepatic metabolism of 2-EHA, (2) the same metabolites were formed in rats and man and (3) an unsaturated terminal olefin, 2-ethyl-5-hexenoic acid is formed in the liver.

Distinction of dicarboxylic aciduria due to medium-chain triglyceride feeding from that due to abnormal fatty acid oxidation and fasting in children.

Increased amounts of dicarboxylic acids are excreted in human urine under conditions of medium-chain triglyceride (MCT) feeding, abnormal fatty acid oxidation (FAO) and fasting. Criteria to distinguish dicarboxylic aciduria originating from MCT feeding and other conditions are needed in urinary organic acid profiling for detecting inborn errors of metabolism. Patterns of dicarboxylic aciduria in children under various conditions were compared. The relative amounts of medium-chain saturated dicarboxylic acids in urine are not reliable for identifying MCT-induced dicarboxylic aciduria. On the other hand, low ratios of unsaturated to saturated dicarboxylic acids (<0.1) and 3- hydroxydecenedioic to 3-hydroxydecanedioic acids were found to be useful in identifying dicarboxylic aciduria due to MCT ingestion. Additional unique features of dicarboxylic aciduria from MCT are low ratios of 3-hydroxydodecanedioic to 3-hydroxydecanedioic acid (<0.14) and 3-hydroxyadipic to adipic acid (<0.02).

Metabolism of 2-ethylhexanol administered orally and dermally to the female Fischer 344 rat.

1. Excretion balance studies were conducted with 2-ethylhexanol (2-EH) in female Fischer 344 rats following single high (500 mg/kg) and low (50 mg/kg) oral doses of [14C]2-EH, following repeated oral dosing with unlabelled 2-EH at the low level, following dermal exposure for 6 h with a 1 g/kg applied dose of [14C]2-EH, and following a 1 mg/kg i.v. dose of [14C]2-EH. 2. The high, low and repeated low oral dose studies with 2-EH showed similar excretion balance profiles of [14C], with some evidence of metabolic saturation at the high dose. 3. No evidence of metabolic induction was seen following the repeated low oral dosing. 4. All of the oral doses were eliminated rapidly, predominantly in the urine during the first 24 h following dosing. 5. The dermal dosing resulted in only about 5% absorption of the 1 g/kg dose, with the major portion of the dose recovered unabsorbed from the dermal exposure cell at 6 h. 6. Urinary metabolites eliminated following the oral and dermal doses were predominantly glucuronides of oxidized metabolites of 2-EH, including glucuronides of 2-ethyladipic acid, 2-ethylhexanoic acid, 5-hydroxy-2-ethylhexanoic acid and 6-hydroxy-2-ethylhexanoic acid.

An assessment of the dietary uptake of di-2-(ethylhexyl) adipate (DEHA) in a limited population study.

The plasticizer di-2-(ethylhexyl) adipate (DEHA), which may be present in food-contact films, can migrate into certain foodstuffs. Results from plasticizer migration studies into food have enabled an indirect estimate of the maximum daily dietary intake of DEHA. A previous study of the metabolism and pharmacokinetics of DEHA in humans identified the urinary metabolite 2-ethylhexanoic acid (EHA) as a useful marker metabolite for assessing DEHA intake. The present study was designed to investigate urinary EHA concentrations following a controlled dose of DEHA presented with food, and to assess the average daily intake of DEHA in a limited population survey. The urinary elimination profile of EHA, following a dose of DEHA in food, showed that in order to extrapolate DEHA intake from EHA measurements, a 24-hr urine sample was required. In the survey the elimination of EHA was determined in 24-hr urine samples in 112 individuals from five different geographical locations in the UK. No restrictions were placed on age or gender. Estimates of daily intake of DEHA show a skewed distribution with a median value of 2.7 mg. This is similar to an estimated maximum daily intake of 8.2 mg/day, derived using an indirect method by the UK Ministry of Agriculture, Fisheries and Food.

Exposure of newborn infants to di-(2-ethylhexyl)-phthalate and 2-ethylhexanoic acid following exchange transfusion with polyvinylchloride catheters.

Infants in the neonatal intensive care unit are regularly exposed to the plasticizer di-(2-ethylhexyl)-phthalate (DEHP) following exchange transfusion or extracorporeal membrane oxygenation. Whether such exposure leads to increased morbidity is not known, although elevated levels of DEHP have been associated with necrotizing enterocolitis and cholestasis. The hypothesis that infants undergoing exchange transfusion are exposed to toxic levels of DEHP and the presumed metabolite 2-ethylhexanoic acid (EHXA) was tested by measuring serum levels of DEHP in 16 newborn infants (gas-liquid-chromatography) and urine concentrations of EHXA in 6 of these infants (gas chromatography-mass spectrometry). DEHP levels were undetectable (< 1 microgram/mL) before exchange but ranged from 6.1 to 21.6 micrograms per mL of serum (average, 12.5 +/- 6.2 micrograms/mL) after a single exchange transfusion. DEHP uptake did not result in cholestasis. EHXA peak levels were 127 to 416 ng per mL of urine, with a median of 174 ng per mL. Concentrations of EHXA were lower than anticipated, which indicates that EHXA is not a major metabolite in the neonatal infant.

Novel glycine conjugates in medium-chain acyl-CoA dehydrogenase deficiency.

The glycine conjugates of isocaproic, 4-methylhexanoic, 7-hydroxyoctanoic and 8-hydroxyoctanoic acids have been identified in the urine of children with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using gas chromatography-mass spectrometry of the trimethylsilyl derivatives. A quantitative study showed that the glycine conjugates of isocaproic and 4-methylhexanoics acids were excreted during acute episodes and in smaller amounts when subjects were asymptomatic. The glycine conjugates of 7-hydroxyoctanoic and 8-hydroxyoctanoic acids were detectable during acute episodes. None of the conjugates was detected in controls or controls receiving a diet containing medium-chain triglycerides. It is suggested that the glycine conjugates of isocaproic acid and 4-methylhexanoic acid are metabolites of branched-chain fatty acids and that they are specific for MCAD deficiency.