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.

Analysis of metabolic profiles of generalized aggressive periodontitis.

BACKGROUND AND OBJECTIVE: The specific pathogenesis of generalized aggressive periodontitis (GAgP) has not yet been clarified, and few studies have focused on the association between GAgP and metabolomics. To elucidate the roles of metabolic profiles in the status of GAgP, this study aimed to identify the differential metabolic profiles between patients with GAgP and healthy controls using an untargeted metabolomic profiling method. MATERIAL AND METHODS: Serum and gingival crevicular fluid samples were collected from healthy controls (n = 20) and patients with GAgP (n = 20) in this cross-sectional study. The relative levels of biomarkers in the samples were measured by gas chromatography-mass spectrometry. Principal components analysis and orthogonal partial least-squares discriminant analysis were used for statistical analysis. Metabolites were analysed qualitatively using the FiehnLib and NIST databases. Full-mouth probing depth and clinical attachment loss were recorded as indexes of periodontal disease. RESULTS: A total of 349 metabolites were qualitatively detected in the gingival crevicular fluid samples, and 200 metabolites were detected in the serum samples. Compared with healthy controls, patients with GAgP showed significant increases in serum urea and allo-inositol levels. In contrast, glutathione, 2,5-dihydroxybenzaldehyde, adipic acid and 2-deoxyguanosine levels were decreased in patients with GAgP. In the gingival crevicular fluid samples, noradrenaline, uridine, α-tocopherol, dehydroascorbic acid, xanthine, galactose, glucose-1-phosphate and ribulose-5-phosphate levels were increased in patients with GAgP, while thymidine, glutathione and ribose-5-phosphate levels were decreased. CONCLUSION: The metabolomics analysis by gas chromatography-mass spectrometry is an effective and minimally non-invasive way to differentiate the metabolites characteristic of patients with GAgP. Both serum and gingival crevicular fluid metabolomics are significantly different between patients with GAgP and healthy controls. These metabolic profiles have great potential in detecting GAgP and helping to understand its underlying mechanisms.

A discovery-driven approach to elucidate urinary metabolome changes after a regular and moderate consumption of beer and nonalcoholic beer in subjects at high cardiovascular risk.

SCOPE: The aim of this work was to study the urinary metabolomics changes of participants that consumed beer, nonalcoholic beer (na-beer), and gin. METHODS AND RESULTS: Thirty-three males at high cardiovascular risk between 55 and 75 years old participated in an open, randomized, crossover, controlled trial with three nutritional interventions consisting of beer, na-beer, and gin for 4 wk. Diet and physical activity was monitored throughout the study and compliance was assessed by measurement of urinary isoxanthohumol. Metabolomic analysis was performed in urine samples by LC coupled to an LTQ-Orbitrap mass spectrometer combined with univariate and multivariate statistical analysis. Ten metabolites were identified. Eight were exogenous metabolites related to beer, na-beer, or gin consumption, but two of them were related to endogenic changes: hydroxyadipic acid linked to fatty acid oxidation, and 4-guanidinobutanoic acid, which correlated with a decrease in urinary creatinine. Plasmatic acylcarnitines were quantified by targeted MS. A regular and moderate consumption of beer and na-beer decreased stearoylcarnitine concentrations. CONCLUSION: Humulinone and 2,3-dihydroxy-3-methylvaleric acid showed to be potential biomarkers of beer and na-beer consumption. Moreover, the results of this trial provide new evidence that the nonalcoholic fraction of beer may increase fatty oxidation.

Simultaneous determination of phthalates and adipates in human serum using gas chromatography-mass spectrometry with solid-phase extraction.

A gas chromatography-mass spectrometry assay was developed and validated for the simultaneous determination of phthalates and adipates in human serum. The phthalates and adipates studied were dimethyl phthalate, diethyl phthalate, dibutyl phthalate, benzylbutyl phthalate, di-2-ethylhexyl phthalate, di-n-octyl phthalate, diethyl adipate, dibutyl adipate, diisobutyl adipate, bis(2-butoxyethyl) adipate and di-2-ethylhexyl adipate, with diisooctyl phthalate as internal standard. The extraction and cleaning up procedure was carried out with solid-phase extraction cartridges containing dimethyl butylamine groups, which showed extraction efficiencies over 88% for each analyte and the internal standard. The calibration curves obtained were linear with correlation coefficients greater than 0.98. For all analytes, the assay gave CV% values for intra-day precision from 4.9 to 13.3% and mean accuracy values from 91.4 to 108.4%, while inter-day precision was 5.2-13.4% and mean accuracy 91.0-110.2%. The limits of detection for the assay of phthalates and adipates were in the range 0.7-4.5 ng/mL. The method is simple, sensitive and accurate, and allows for simultaneous determination of nanogram levels of phthalates and adipates in human serum. It was successfully applied to an investigation on the level of phthalates and adipates in a non-occupationally exposed population.

[Metabonomic profiling of plasma metabolites in Wistar rats to study the effect of aging by means of GC/TOFMS-based techniques].

The global metabolite profiles of endogenous compounds of Wistar rats from 12 to 20 weeks old were investigated to take deep insight into and get better understanding of the pathogenesis of development and aging. Plasma from Wistar rats at 12, 14, 16, 18, and 20 weeks old were analyzed using GC/TOFMS. Multivariate data analysis was then used to process the metabonomic data which indicated excellent separation between different weeks and showed that the metabolic profiles of the samples changed with age, enabling age-related metabolic trajectories to be visualized. Decreased concentrations of citric acid, cis-aconitic acid, 9-(z)-hexadecenoic acid along with increased levels of hexanedioic acid, alpha-tocopherol, 3-indole propionic acid, etc contributed to the separation. Several major metabolic pathways were identified to be involved in metabolic regulation. This suggests that GC/TOFMS-based metabonomics is a powerful alternative approach to identifying potential biomarkers and investigating the physiological developments of aging and it is important to employ suitable age-match control group in metabonomic study of physiological monitoring, drug safety assessment, and disease diagnosis, etc.

Preparation of samples for gas chromatography/mass spectrometry analysis of phthalate and adipate esters in plasma and beverages by steam distillation and extraction.

Phthalate and adipate esters are present in relatively large amounts in the environment, resulting in their large blank values at analysis and making precise analysis difficult. We developed a highly sensitive analytical method for phthalate and adipate esters in plasma and beverages by lowering the blank values that interfere with analysis. The method uses a closed distillation cleanup system in which steam distillation and extraction are performed simultaneously. The recoveries from beverages and plasma were both satisfactory, ranging from 90.2 to 118.3%, relative standard deviation (RSD) = 2.8-5.3%, and 96.2-134.4%, RSD = 2.2-6.5%, respectively. The detection limits of dibutyl phthalate and di-2-ethyl hexyl phthalate were 5 ng/mL, and those of diethyl phthalate, butyl benzyl phthalate, and di-2-ethyl hexyl adipate were 10 ng/mL in rabbit plasma and beverages.

Gas chromatographic-mass spectrometric screening for organic acidemias using dried urine filter paper: determination of alpha-ketoacids.

There are several organic acid disorders that require information on alpha-ketoacids, such as maple syrup urine disease or alpha-ketoadipic acidemia. The recovery, stability and diagnostic availability of alpha-ketoacids in dried urine filter paper analyzed by GC-MS with oxime-trimethylsilyl derivatization was studied for organic acidemia screening. The recovery of all nine types of alpha-ketoacids tested, but for phenylpyruvate, 2-ketoadipate, and p-OH-phenylpyruvate, from filter paper samples was acceptable. The stability of pyruvate, branched-chain alpha-ketoacids, alpha-ketoadipate and alpha-ketoglutarate was stable for at least 28 days, although some alpha-ketoacids such as succinylacetone were unstable. It indicated it was difficult to diagnose only tyrosinemia type 1 among nine specimens from organic acidemia patients tested. The method could be applied to global organic acidemia screening.

[Polyphenolic antioxidants in fruit juice. Urinary excretion and effects on biological markers for antioxidative status]. / Polyfenoliske antioxidanter i frugtjuice. Udskillelse i urin samt indflydelse på biomarkører for antioxidativ status.

This intervention study was designed as cross-over (four women, one man) with three doses of black currant/apple (1:1) juice (750, 1000, and 1500 mL) for one week corresponding to an intake of 4.8, 6.4, and 9.6 mg quercetin per day. Urinary excretion of quercetin increased significantly with dose and with time. The fraction excreted in urine was constant 0.29-0.47%. Plasma quercetin did not change with juice intervention. Plasma ascorbate increased during intervention due to ascorbate from the juice. Total plasma malondialdehyde decreased with time during 1500 mL juice intervention. Plasma protein 2-adipic semialdehyde residues, increased with time and dose, and glutathione peroxidase increased with juice dose, whereas other selected markers of oxidative status did not change. These effects might be related to several components of the juice and cannot be attributed solely to its quercetin content.

Kearns-Sayre syndrome presenting as 2-oxoadipic aciduria.

A patient with 2-oxoadipic aciduria and 2-aminoadipic aciduria presented at 2 years of age with manifestations typical of organic acidemia, episodes of ketosis and acidosis, progressive to coma. This resolved and the key metabolites disappeared from the urine and blood. At 9 years of age she developed typical Kearns-Sayre syndrome with complete heart block, retinopathy, and ophthalmoplegia. Southern blot revealed a deletion in the mitochondrial genome.

Determination of glutamine and alpha-ketoglutarate concentration and specific activity in plasma using high-performance liquid chromatography.

A method is described for measuring glutamine (GLN) and alpha-ketoglutarate (KG) concentration and specific activity (SA) using high-performance liquid chromatography (HPLC). Plasma GLN and KG are separated on miniature ion-exchange columns. KG is derivatized with O-phenylene diamine, the derivative is extracted in ethyl acetate, dried, and dissolved in pH 7 phosphate buffer. The isolated GLN is enzymatically converted to KG and analysed as such. Derivatized samples are stable for weeks at -20 degrees C. Samples are injected onto a reversed-phase HPLC column. Absolute standards are injected to determine the nmol content of unknown samples. alpha-Ketoadipate and [3H]-glutamine are used as internal standards to quantitate KG and GLN concentrations, respectively. Collection of the entire peak of interest permits determination of the radioactivity in the GLN and KG peaks; this together with the determination of the nanomoles injected permits the calculation of the SA. Typical precision is 3.5 and 4.6% for GLN and KG concentrations and 5.3 and 3.3% for GLN and KG SA, respectively. Analysis time is ca. 7 min. Using this method, the turnover rate of GLN carbon was determined during a 5-h infusion of L-[U-14C]glutamine in a human subject.

Recurrent, familial Reye-like syndrome with a new complex amino and organic aciduria.

Five of 13 siblings from a Jewish-Ashkenazi family suffered from recurrent Reye-like episodes. During attacks, these patients excreted alpha-keto-adipic, alpha-hydroxy-adipic, and alpha-aminoadipic acids, branched-chain keto acids and saccharopine in addition to lactic, pyruvic, and dicarboxylic acids characteristic of Reye syndrome. The serum concentrations of citrulline and alpha-amino-adipic acid were elevated and carnitine was at the upper limit of the normal range. Serum acetoacetate level was 4-5 times the beta-hydroxybutyrate level, but the pyruvate/lactate ratio was normal. Notably, plasma ketone bodies were lower than expected from the degree of catabolism. When the patients were symptom-free, no abnormal amino or organic acids in serum or urine were detected. These findings might be interpreted as a functional impairment at three different biochemical sites: fatty acid beta-oxidation, dehydrogenase complexes of the pyruvic, alpha-ketoglutaric, alpha-ketoadipic, and branched-chain keto acids, and pyruvate carboxylase. We suggest that in this hereditary disorder a toxic substance, exogenously or endogenously derived, interfered at multiple sites in different metabolic pathways.

3-Hydroxy-3-methylglutaric, adipic, and 2-oxoglutaric acids measured by HPLC in the plasma from diabetic patients.

A method for the measurement of organic acids in human plasma is presented. The analytical procedure consists of plasma protein precipitation with acetonitrile, acid extraction by chromatography through a DEAE-cellulose column eluted with 100 mM perchloric acid, HPLC by cation-exchange column Aminex HPX-87 eluted with 6.5 mM sulfuric acid. Adipic, 3-hydroxy-3-methylglutaric, 2-oxoglutaric, and citric acids were determined in the plasma of diabetic patients. The concentrations of all the measured acids, but particularly those of adipic and 3-hydroxy-3-methylglutaric acids, were significantly higher than those of healthy controls. These results suggest that in diabetics the omega-oxidation of fatty acids is enhanced.

Pattern of aliphatic dicarboxylic acids in uremic serum including a new organic acid, 2,4-dimethyladipic acid.

(1) 2,4-Dimethyladipic acid was first identified in normal human urine using gas chromatography-mass spectrometry. Urinary excretion of 2,4-dimethyladipic acid in 7 healthy adults ranged from 4.9 mumol to 14 mumol per 24 h. (2) Succinic acid, adipic acid, 3-methyladipic acid, 2,4-dimethyladipic acid, pimelic acid and azelaic acid were identified in the ultrafiltrate of the blood obtained from a chronic uremic patient using a hemodialyzer. (3) Levels of succinic acid, adipic acid, 3-methyladipic acid, 2,4-dimethyladipic acid, pimelic acid and azelaic acid in uremic serum were determined using a mass fragmentographic technique. Concentration of succinic acid in uremic serum was comparable to that in normal serum, whereas concentrations of adipic acid, 3-methyladipic acid, 2,4-dimethyladipic acid, pimelic acid and azelaic acid were highly elevated in uremic serum.

The release of plasticizer from polyvinyl chloride haemodialysis tubing.

The extent of migration of plasticizer from haemodialysis lines into saline and plasma has been examined and a number of different types of tubing have been compared. A model system designed to simulate in vivo haemodialysis was evolved. The method uses a continuous cyclohexane extraction in order to mimic uptake of plasticizer by body tissues. Although not an ideal system, the results obtained are indicative of the extent to which the plasticizers are leached.

Alpha-ketoadipic aciduria, a new inborn error of lysine metabolism; biochemical studies.

Investigation of a psychomotorically retarded girl showed excretion of abnormal amounts of alpha-ketoadipic acid, alpha-hydroxyadipic acid, alpha-aminoadipic acid, 1,2-butenedicarboxylic acid and elevation of plasma alpha-aminoadipic acid levels. The identity of these metabolities was established by various methods. The excretion of alpha-aminoadipic acid correlated to the lysine intake. Degradation studies with cultured fibroblasts indicate a defect in the oxidative decarboxylation of alpha-ketoadipic acid (see Clin. Chim. Acta, 58 (1975) 271.

In vivo studies on the metabolism of hexanedioic acid.

1. Using the combined gas-liquid chromatography-mass spectrometry technique it was shown that ketotic patients excreted up to 273 mg of hexanedioic acid daily in their urine, whereas serum samples from these patients contained only trace amounts of this acid. Healthy humans excreted 2-5 mg daily. Hexanedioic acid was not detectable in normal serum. 2. An experiment with the infusion of large amounts of 3-hydroxybutyrate into a dog indicated that the increased urinary hexanedioic acid excretion in ketosis is not due to a competition between 3-hydroxybutyrate and hexanedioic acid for the same renal reabsorption mechanism. 3. [ 1,6-14-C]Hexanedioic acid intravenously injected into a dog was at first distributed in the extracellular space, followed by a partial equilibration with the intracellular space. About 11% of the injected dose was expired as 14-CO2 in 220 min. The maximal 14-CO2 production rate was obtained after about 20 min. In 240 min, 47% of the injected radioactivity was recovered in the urine. The large urinary excretion of labeled hexanedioic acid observed in the presence of only trace amounts in serum, showed that the high excretion by ketotic patients of the dicarboxylic acid may be explained without postulating an exclusive renal synthesis for hexanedioic acid.