Plasticizer exposure of infants during cardiac surgery.

In the present study we investigated the internal exposure situation of infant patients to the plasticizers TEHTM (tri-2-ethylhexyl trimellitate) and DEHP (di-2-ethylhexyl phthalate). The study collective included 21 infant patients aged 2-22 months that had to undergo cardiac surgery using cardio pulmonary bypass (CPB). Each patient, but one, received blood products during surgery. A special feature was that the used CPB tubings were exclusively plasticized with the alternative plasticizer TEHTM and were free of the standard plasticizer DEHP, that raises increasing toxicological concern. The blood products were stored in DEHP plasticized blood bags. Blood and urine samples of each infant patient were analysed before and after the surgery for the levels of the plasticizers DEHP and TEHTM and their metabolites. In general, the plasticizers were detected in the post-surgery blood samples only, with TEHTM in low levels (median 18.4 µg/L) and DEHP in rather elevated levels (median 1046 µg/L). With respect to the urine samples, TEHTM metabolites were not detected in any of the samples. DEHP metabolites were found in all urine samples, however, in significantly increased median levels in the post-surgery urine samples of the infants (increase factor 5-26). Thus, the present study clearly demonstrates the strong contribution of standard medical procedures to the internal plasticizer burden of patients. Particularly with regard to the suspected endocrine disrupting activities of the phthalate plasticizer DEHP, the elevated internal levels of this plasticizer and its metabolites in infants following cardiac surgery are alarming.

Reduction of exposure to plasticizers in stored red blood cell units.

INTRODUCTION: Plastic can be toxic and hazardous to an organism's health, but it is being widely used in our daily lives. Di-2-ethylhexyl-phthalate is the most common plasticizer in medical devices made of polyvinylchloride and is commonly found in soft bags storing red blood cell units. Di-2-ethylhexyl-phthalate and its degradation product mono-2-ethylhexyl-phthalate can migrate into human body fluids, for example, blood and tissues. The aim of the study was to assess the concentration of plasticizers in red blood cell units according to storage time and after mechanical rinsing using a cell salvage device. METHODS: Levels of di-2-ethylhexyl-phthalate and mono-2-ethylhexyl-phthalate were analysed in 50 unwashed red blood cell units using liquid chromatography coupled with tandem mass spectrometry. In addition, phthalate concentrations were measured before and after mechanical rinsing in six more washed red blood cell units with storage times ranging between 36 and 56 days. A linear regression model was determined by the daily increase of di-2-ethylhexyl-phthalate and mono-2-ethylhexyl-phthalate in the stored red blood cell units subject to their storage time (range = 4-38 days), and the effect of mechanical rinsing on their phthalate concentration was calculated. RESULTS: A linear correlation was found between storage time of unwashed red blood cell units and the concentration of di-2-ethylhexyl-phthalate (p < 0.001) or mono-2-ethylhexyl-phthalate (p < 0.001). Stored red blood cell units older than 14 days had significantly higher concentrations of both contaminants than red blood cell units of shorter storage time (p < 0.001). Mechanical rinsing in washed red blood cell units attained a reduction in the di-2-ethylhexyl-phthalate and mono-2-ethylhexyl-phthalate concentration by a median of 53% (range = 18-68%; p = 0.031) and 87% (range = 68-96%; p = 0.031), respectively. CONCLUSION: Leaching of di-2-ethylhexyl-phthalate and mono-2-ethylhexyl-phthalate into red blood cell units depends on the duration of storage time. Plasticizers can be significantly reduced by mechanical rinsing using cell salvage devices, and thus, red blood cell units can be regenerated with respect to chemical contamination.

Human metabolism and kinetics of tri-(2-ethylhexyl) trimellitate (TEHTM) after oral administration.

Tri-(2-ethylhexyl) trimellitate (TEHTM) is a plasticizer for PVC material and is used for medical devices as an alternative to di-(2-ethylhexyl) phthalate. As plasticizers are known to migrate easily into contact liquids, exposure of patients to TEHTM is highly probable. In the present study, human metabolism pathways of TEHTM and its elimination kinetics were investigated. For that purpose, four healthy volunteers were orally exposed to a single dose of TEHTM. TEHTM and its postulated primary metabolites were investigated in blood samples (up to 48 h after exposure), and in urine samples (collected until 72 h after exposure) using liquid chromatography tandem mass spectrometry (LC-MS/MS). TEHTM was found to be regioselectively hydrolyzed to its diesters di-2-(ethylhexyl) trimellitates (1,2-DEHTM, 2,4-DEHTM) with maximum blood concentrations at 3-h post-exposure, and to its monoester isomers mono-2-(ethylhexyl) trimellitates (1-MEHTM, 2-MEHTM) with peak blood concentrations 5-h post-exposure. For the elimination of investigated urinary metabolites, biphasic elimination kinetics was observed. The most dominant urinary biomarker was found to be 2-MEHTM (2-mono-(2-ethylhexyl) trimellitate), followed by several specific secondary metabolites. All in all, approximately 5.8% of the orally administered dose was recovered in urine over a period of 72 h, indicating a comparatively low resorption rate of TEHTM in humans in combination with an apparently rather slow metabolism and excretion rate. In fact, TEHTM and selected metabolites were still detectable in blood and urine 48-h and 72-h post-exposure, respectively. This study is the first to elucidate TEHTM metabolism pathways in humans and to identify metabolites of TEHTM in blood and urine by usage of especially designed human biomonitoring methods. Powerful tools for exposure monitoring and risk assessment of TEHTM are therewith available for future research.

Effect of phospholipid coating on the migration of plasticizers from PVC tubes.

Plasticizers in polyvinyl chloride (PVC) are not covalently bound to the polymer and can thus migrate into the contact medium. The presented study investigated the potential effects of phospholipid-lining as anti-coagulation coating (ACC) on the migration rate of plasticizers from PVC tubing into blood. For the in-vitro study, five different groups of tubing sets in six replicates were perfused with sheep blood (Group A: PVC plasticized with di-(2-ethylhexyl) phthalate (DEHP) without ACC, Group B: DEHP-plasticized PVC with ACC, Group C: PVC plasticized with tri-(2-ethylhexyl) trimellitate (TOTM) without ACC, Group D: TOTM-plasticized PVC with ACC, Group E (control group): polyolefin material with ACC but without plasticizers). Both the levels of the unchanged plasticizers in blood and the concentration levels of their primary degradation products were assessed. For DEHP, the primary metabolite MEHP (mono-(2-ethylhexyl) phthalate) was determined. The isomers of MEHTM (mono-(2-ethylhexyl) trimellitate) and DEHTM (di-(2-ethylhexyl) trimellitate), respectively, were investigated as primary metabolites of TOTM. The calculated DEHP equivalents (sum of determined levels of DEHP and MEHP) after 24 h of perfusion displayed a tendency towards lower levels in the tubing sets without ACC (Group A (201 ±â€¯56.4 µmol/L)) compared to the tubing sets with ACC (Group B (253 ±â€¯369 µmol/L)). Significantly different DEHP equivalents between Group A and Group B were found after a perfusion time of 6 h and 10 h, respectively. A similar effect was observed for the TOTM-containing tubing sets. However, the absolute plasticizer migration rate of TOTM (TOTM equivalents) after 24 h of perfusion was found to be significantly lower compared to that of DEHP (with a factor of over 200). The results indicate that phospholipid coating (ACC) rather enhances the migration of plasticizers and of their primary degradation products from PVC tubing into streaming blood. The enhancement effect was found to be slightly greater for TOTM, but as TOTM migrates in significantly lower levels than DEHP in all experimental settings, TOTM is confirmed to be a recommendable alternative plasticizer to DEHP in medical devices.

Comprehensive monitoring of specific metabolites of tri-(2-ethylhexyl) trimellitate (TEHTM) in urine by column-switching liquid chromatography-tandem mass spectrometry.

Tri-(2-ethylhexyl) trimellitate (TOTM or TEHTM) is a substitute for the plasticizer di-(2-ethylhexyl) phthalate (DEHP). Here, a fast and robust HPLC method is presented for the first time enabling the simultaneous quantification of several TEHTM metabolites in urine. These are the three TEHTM monoester isomers 1-mono-(2-ethylhexyl) trimellitate (1-MEHTM), 2-mono-(2-ethylhexyl) trimellitate (2-MEHTM), and 4-mono-(2-ethylhexyl) trimellitate (4-MEHTM) as well as several selected side chain oxidized monoesters of TEHTM, namely, 1-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-1-MEHTM), 2-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-2-MEHTM), 1-mono-(2-ethyl-5-oxohexyl) trimellitate (5oxo-1-MEHTM), 2-mono-(2-ethyl-5-oxohexyl) trimellitate (5oxo-2-MEHTM), 1-mono-(2-ethyl-5-carboxypentyl) trimellitate (5cx-1-MEPTM), 2-mono-(2-ethyl-5-carboxypentyl) trimellitate (5cx-2-MEPTM), 2-mono-(2-carboxymethylhexyl) trimellitate (2cx-2-MMHTM), and 1-mono-(2-carboxymethylhexyl) trimellitate (2cx-1-MMHTM). The method is characterized by a short sample preparation, for which the urine samples are enzymatically hydrolyzed and cleaned up by an online column arrangement. Separation of the analytes is enabled using liquid chromatography coupled with tandem mass spectrometry. Thus, in less than 30 min, 11 postulated metabolites of TEHTM can be selectively and sensitively quantified. The method is distinguished by its wide linear working range of up to 1800 µg/L with detection limits ranging from 0.3 µg/L (for 5oxo-1-MEHTM) to 1.5 µg/L (for 1-MEHTM). Precision and repeatability of the method were proven with determined relative standard deviations between 2.5 and 11.3%. The selection of the analytes of this method was based on a pilot study, by which several potential TEHTM metabolites were investigated in human urine of an orally exposed volunteer. Thus, the here presented method is a perfect tool for human biomonitoring of TEHTM exposure. Graphical abstract Analysis of several postulated TEHTM metabolites in human urine using LC-MS/MS.

Regioselective ester cleavage of di-(2-ethylhexyl) trimellitates by porcine liver esterase.

In a comparative study the ester hydrolysis of the plasticizers di-(2-ethylhexyl) phthalate (DEHP) and tri-(2-ethylhexyl) trimellitate (TEHTM) as well as of the diester isomers 1,2-di-(2-ethylhexyl) trimellitate (1,2-DEHTM), 1,4-di-(2-ethylhexyl) trimellitate (1,4-DEHTM) and 2,4-di-(2-ethylhexyl) trimellitate (2,4-DEHTM) was investigated by a newly developed in vitro experimental design using porcine liver esterase (PLE). The substrates were incubated with PLE for 48h at 25°C in borate buffer and samples were taken at predetermined intervals during the experiment. The samples were processed using liquid-liquid extraction and analyzed using LC-MS/MS. The results demonstrated a rapid and extensive hydrolysis of the diester DEHP to the monoester mono-(2-ethylhexyl) phthalate (MEHP) during the incubation with PLE. The isomers of DEHTM were also hydrolyzed by PLE to a high extent, whereas TEHTM showed a high stability against enzymatic hydrolysis. The regioselective analysis revealed that the monoester isomers 1-MEHTM and 2-MEHTM were predominantly produced during the degradation of DEHTM isomers, indicating a preferred hydrolysis at the para-position. These findings are eminent for planning further investigations on the human TEHTM metabolism, as the extent, rate and route of metabolism are of crucial importance for a toxicological assessment.

Isomeric separation and quantitation of di-(2-ethylhexyl) trimellitates and mono-(2-ethylhexyl) trimellitates in blood by LC-MS/MS.

A new and fast HPLC-method for the simultaneous determination of tri-(2-ethylhexyl) trimellitate (TOTM or TEHTM), its diesters 2,4-di-(2-ethylhexyl) trimellitate (2,4-DEHTM), 1,4-di-(2-ethylhexyl) trimellitate (1,4-DEHTM), 1,2-di-(2-ethylhexyl) trimellitate (1,2-DEHTM) and monoesters 1-mono-(2-ethylhexyl) trimellitate (1-MEHTM), 2-mono-(2-ethylhexyl) trimellitate (2-MEHTM) and 4-mono-(2-ethylhexyl) trimellitate (4-MEHTM) together with di-(2-ethylhexyl) phthalate (DEHP) and its primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) in blood was developed and validated. The analytes are extracted from blood using liquid-liquid extraction and are chromatographically separated by reversed-phase HPLC using core shell material. Quantitative assessment was performed by ESI-tandem mass spectrometry in negative ionization mode using stable isotope dilution. In less than 30min six postulated primary metabolites of TOTM along with the DEHP metabolite MEHP can be selectively and sensitively quantified. Additionally, the method enables the determination of the parent plasticizers TOTM and DEHP. The detection limits in blood were found to range between 0.7-5.5µg/L for all TOTM analytes. Precision and repeatability of the method were proven by relative standard deviations between 0.9% and 8.7%. TOTM, an alternative plasticizer to DEHP, is already increasingly used for medical devices. Nevertheless, data about the human metabolism of TOTM are still limited. The presented method is the first one enabling the simultaneous determination of the parent plasticizers TOTM and DEHP together with their primary degradation products (DEHTM, MEHTM, MEHP) and can thus be applied manifold including the investigation of the human metabolism of TOTM.