Characterizing concentrations of diethylene glycol and suspected metabolites in human serum, urine, and cerebrospinal fluid samples from the Panama DEG mass poisoning.

CONTEXT: Diethylene glycol (DEG) mass poisoning is a persistent public health problem. Unfortunately, there are no human biological data on DEG and its suspected metabolites in poisoning. If present and associated with poisoning, the evidence for use of traditional therapies such as fomepizole and/or hemodialysis would be much stronger. OBJECTIVE: To characterize DEG and its metabolites in stored serum, urine, and cerebrospinal fluid (CSF) specimens obtained from human DEG poisoning victims enrolled in a 2006 case-control study. METHODS: In the 2006 study, biological samples from persons enrolled in a case-control study (42 cases with new-onset, unexplained AKI and 140 age-, sex-, and admission date-matched controls without AKI) were collected and shipped to the Centers for Disease Control and Prevention (CDC) in Atlanta for various analyses and were then frozen in storage. For this study, when sufficient volume of the original specimen remained, the following analytes were quantitatively measured in serum, urine, and CSF: DEG, 2-hydroxyethoxyacetic acid (HEAA), diglycolic acid, ethylene glycol, glycolic acid, and oxalic acid. Analytes were measured using low resolution GC/MS, descriptive statistics calculated and case results compared with controls when appropriate. Specimens were de-identified so previously collected demographic, exposure, and health data were not available. The Wilcoxon Rank Sum test (with exact p-values) and bivariable exact logistic regression were used in SAS v9.2 for data analysis. RESULTS: The following samples were analyzed: serum, 20 case, and 20 controls; urine, 11 case and 22 controls; and CSF, 11 samples from 10 cases and no controls. Diglycolic acid was detected in all case serum samples (median, 40.7 mcg/mL; range, 22.6-75.2) and no controls, and in all case urine samples (median, 28.7 mcg/mL; range, 14-118.4) and only five (23%) controls (median, < Lower Limit of Quantitation (LLQ); range, < LLQ-43.3 mcg/mL). Significant differences and associations were identified between case status and the following: 1) serum oxalic acid and serum HEAA (both OR = 14.6; 95% C I = 2.8-100.9); 2) serum diglycolic acid and urine diglycolic acid (both OR > 999; exact p < 0.0001); and 3) urinary glycolic acid (OR = 0.057; 95% C I = 0.001-0.55). Two CSF sample results were excluded and two from the same case were averaged, yielding eight samples from eight cases. Diglycolic acid was detected in seven (88%) of case CSF samples (median, 2.03 mcg/mL; range, < LLQ, 7.47). DISCUSSION: Significantly elevated HEAA (serum) and diglycolic acid (serum and urine) concentrations were identified among cases, which is consistent with animal data. Low urinary glycolic acid concentrations in cases may have been due to concurrent AKI. Although serum glycolic concentrations among cases may have initially increased, further metabolism to oxalic acid may have occurred thereby explaining the similar glycolic acid concentrations in cases and controls. The increased serum oxalic acid concentration results in cases versus controls are consistent with this hypothesis. CONCLUSION: Diglycolic acid is associated with human DEG poisoning and may be a biomarker for poisoning. These findings add to animal data suggesting a possible role for traditional antidotal therapies. The detection of HEAA and diglycolic acid in the CSF of cases suggests a possible association with signs and symptoms of DEG-associated neurotoxicity. Further work characterizing the pathophysiology of DEG-associated neurotoxicity and the role of traditional toxic alcohol therapies such as fomepizole and hemodialysis is needed.

Cholinesterase inhibition and toxicokinetics in immature and adult rats after acute or repeated exposures to chlorpyrifos or chlorpyrifos-oxon.

The effect of age or dose regimen on cholinesterase inhibition (ChEI) from chlorpyrifos (CPF) or CPF-oxon (CPFO) was studied in Crl:CD(SD) rats. Rats were exposed to CPF by gavage in corn oil, rat milk (pups), or in the diet (adults) or to CPFO by gavage in corn oil. Blood CPF/CPFO levels were measured. With acute exposure, ChEI NOELs were 2 mg/kg CPF for brain and 0.5 mg/kg CPF for red blood cells (RBCs) in both age groups. In pups, ChEI and blood CPF levels were similar using either milk or corn oil vehicles. Compared to gavage, adults given dietary CPF (12 h exposure) had greater RBC ChEI, but lower brain ChEI at corresponding CPF doses, indicating an effect of dose rate. With repeated CPF exposures, ChEI NOELs were the same across ages (0.5 and 0.1 mg/kg/day for brain and RBCs, respectively). With CPFO dosing, the ChEI NOELs were 0.1 mg/kg (acute) and 0.01 mg/kg/day (repeated doses) for RBCs with no ChEI in brain at CPFO doses up to 0.5 (pup) or 10 mg/kg (adult) for acute dosing or 0.5 mg/kg/day for both ages with repeat dosing. Thus, there were no age-dependent differences in CPF ChEI via acute or repeated exposures. Pups had less ChEI than adults at comparable blood CPF levels. Oral CPFO resulted in substantial RBC ChEI, but no brain ChEI, indicating no CPFO systemic bioavailability to peripheral tissues.

Simultaneous quantitation of testosterone and estradiol in human cell line (H295R) by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry.

The possible interaction of environmental contaminants with the endocrine system has been an environmental concern since the early 1990s. To examine these interactions test guidelines have been introduced by regulatory agencies to screen for possible endocrine active compounds. One of these guidelines is the EPA's OPPTS 890.1550 [Steroidogenesis (Human Cell Line-H295R)]. This guideline requires the quantification of two major biomarkers (testosterone and estradiol) in various biological test systems. Traditional quantitation methodologies such as Radioimmunoassay (RIA) and Enzyme-linked Immunosorbent Assay (ELISA) have been used to quantify low levels of steroids. However, those methodologies have drawbacks such as the radioactive safety, antibody availability, separate assay for each biomarker, and lack of selectivity. In the current study, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry method (LC/APPI-MS/MS) has been developed and validated for the simultaneous quantitation of testosterone and estradiol in the H295R cell line. Briefly, the media from cultured cells was extracted with dichloromethane (CH(2)Cl(2)) containing internal standards of both testosterone-d(3) and estradiol-(13)C(3); then, the extracted organic layer was concentrated down to dryness. The final residue was derivatized with dansyl chloride solution, and directly analyzed by LC/APPI-MS/MS. The calibration curves, with concentration ranging from 10 to 2500 pg/mL, were linear with coefficient >0.99. The lower limits of quantitation for both testosterone and estradiol were 10 pg/mL. This method was successfully validated to support requirements of the current EPA Steroidogenesis guideline. This type of method may also provide value for rapid and precise measurements of these two hormones in other in vitro or in vivo test systems.