Oxidative stress and biochemical indicators in blood of patients addicted to alcohol treated for acute ethylene glycol poisoning.

Ethylene glycol (EG), in addition to its neurotoxic and nephrotoxic effects, evokes oxidative stress. The aim of this study was to assess the influence of the ethylene glycol on the biochemical indicators and oxidoreductive balance of patients treated for acute poisoning. The total study group consisted of 56 persons including 26 alcoholics who took EG as a substitute for ethyl alcohol in the course of alcohol dependence syndrome and 30 controls. Severity of poisoning, results of acid-base parameters, biochemical, and toxicological tests as well as biomarkers of the oxidative stress in blood were analyzed during the patients' hospitalization. The key issue was to assess the oxidative stress and biochemical disturbances caused by EG and the type of treatment applied in the course of poisoning. Significant changes in some parameters were found both at time of diagnosis and after treatment initiation (ethanol as an antidote and hemodialysis). The most important differences included the activity of hepatic parameters (aspartate aminotransferase, AST) and oxidative stress markers like catalase (CAT); correlation of the lipid peroxidation products level (TBARS) with urea concentration has been shown. On the last day of the hospitalization, in some cases, the mutual correlation between the evaluated markers were observed, for example, between alanine transaminase (ALT) and glutathione reductase (GR), and urea concentration and glutathione level (GSH/GSSG). The concentration of ions (H+) had a major impact on the oxidoreductive balance, correlating with the elevated GR and GSH/GSSG levels.

Liquid Chromatography-Tandem Mass Spectrometric Analysis of Octaethylene Glycol Monodecyl Ether in Rat Plasma and its Application to Pharmacokinetic Studies.

Alcohol ethoxylates (AEs) are a major class of non-ionic surfactants, which are widely used in household, institutional and industrial cleaners, and they are considered as an alternative of nonylphenol. In this study, a rapid, sensitive and reliable bioanalytical method was developed for the determination of octaethylene glycol monodecyl ether (C10E8, an AE) in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Chromatographic separation was performed on a reversed-phase C18 column (2.1 mm × 50 mm, 2.1 µm). The mobile phase consisted of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile (40:60% v/v). The flow rate was 0.3 mL/min. For mass spectrometric detection, the multiple reaction monitoring (MRM) mode was used; the MRM transitions were m/z 511.5 → m/z 133.1 for C10E8 and m/z 423.3 → m/z 133.1 for hexaethylene glycol monodecyl ether (internal standard) in the positive ion mode. A calibration curve was constructed within the range of 2-2,000 ng/mL; the intra- (n = 5) and inter-day (n = 3) precision and accuracy were within 10%. The LC-MS-MS method was specific, accurate and reproducible, and this method was successfully applied in a pharmacokinetic study of C10E8 in rats. C10E8 was intravenously (1 mg/kg, n = 6) and orally (10 mg/kg, n = 7) administered to rats. The kinetic parameters were analyzed based on a noncompartmental statistical model using the pharmacokinetic modeling software (WinNonlin). The oral bioavailability of C10E8 was 34.4%.

Five-year review of a UK 24 hour testing service for plasma ethylene glycol and diethylene glycol.

BACKGROUND: We present a 5-year review of our UK service for plasma ethylene glycol and diethylene glycol determination in cases of acute poisoning. METHODS: Ethylene glycol and diethylene glycol have been measured on all samples received for screening for toxicity by gas chromatography-flame ionization detection over a five-year period. A detailed audit of the results has been undertaken. RESULTS: In this period, we received 811 requests, 56% were for first-time screening and 44% repeat analysis where a positive sample has already been received. Of the first-time screen samples, 33.5% screened positive for glycol poisoning. The mean positive ethylene glycol concentration was 1204 mg/L (range 31 to 8666 mg/L). Diethylene glycol was present in 14% of ethylene glycol positive samples but never found alone. CONCLUSIONS: The data presented here suggest it is not essential to measure diethylene glycol since its inclusion is rarely likely to change patient management.

Validated gas chromatographic-mass spectrometric assay for determination of the antifreezes ethylene glycol and diethylene glycol in human plasma after microwave-assisted pivalylation.

A gas chromatographic-mass spectrometric assay is described for identification and quantification of the antifreezes ethylene glycol (EG) and diethylene glycol (DEG) in plasma for early diagnosis of a glycol intoxication. After addition of 1,3-propanediol as internal standard, the plasma sample was deproteinized by acetone and an aliquot of the supernatant was evaporated followed by microwave-assisted pivalylation. After gas chromatographic separation, the glycols were first identified by comparison of the full mass spectra with reference spectra and then quantified. The quantification has been validated according to the criteria established by the Journal of Chromatography B. The assay was found to be selective. The calibration curves for EG and DEG were linear from 0.1 g/l to 1.0 g/l. The limit of detection for EG and DEG was 0.01 g/l and the limit of quantification for both was 0.1 g/l. The absolute recoveries were 50 and 65% for the low quality control samples and 51 and 73% for the high quality control samples of EG and DEG, respectively. Intra- and inter-day accuracy and precision were inside the required limits. The glycols in frozen plasma samples were stable for more than 6 months. The method was successfully applied to several authentic plasma samples from patients intoxicated with glycols. It has also been suitable for analysis of EG and DEG in urine.

Simultaneous detection and quantitation of diethylene glycol, ethylene glycol, and the toxic alcohols in serum using capillary column gas chromatography.

Determination of toxic glycols and alcohols in an emergency setting requires a rapid yet accurate and reliable method. To simultaneously determine diethylene glycol (DEG) along with ethylene glycol, methanol, isopropanol, acetone, and ethanol, we modified a previously developed gas chromatographic (GC) method. The system used a Hewlett-Packard 6890 GC with EPC, a Gooseneck splitless liner, and an Rtx-200 capillary column (30 m x 0.53-mm i.d., 3 mm). After serum samples were deproteinized using ultrafiltration (Millipore Ultrafree-MC), 1 mL of the protein-free filtrate was manually injected into the GC. Internal standards for alcohols (and acetone) and glycols were n-propanol and 1,3-butanediol, respectively. All compounds eluted within 3.5 min (linear temperature gradient from 40 to 260 degrees C); total run time was 6.5 min. Limit of detection and linear range for all compounds were 1 or 2.5 mg/dL and 0-500 mg/dL, respectively. In addition, there was no interference from propionic acid, propylene glycol, and 2,3-butanediol. The modifications in the equipment and temperature program allowed increased resolution and thus, detection and reliable quantitation of DEG and other common toxic glycols and alcohols of clinical interest.

Haematological and spermatotoxic effects of ethylene glycol monomethyl ether in copper clad laminate factories.

OBJECTIVES: To investigate the effects of ethylene glycol monomethyl ether (EGME) on haematology and reproduction in exposed workers. METHODS: 53 Impregnation workers from two factories that make copper clad laminate with EGME as a solvent were recruited as the exposed group. Another group of 121 lamination workers with indirect exposure to EGME was recruited as the control group. Environmental monitoring of concentrations of EGME in air and biological monitoring of urinary methoxyacetic acid (MAA) concentrations were performed. Venous blood was collected for routine and biochemical analyses. Semen was collected from 14 workers exposed to EGME for sperm analysis and was compared with 13 control workers. RESULTS: Results of haematological examination showed that the haemoglobin, packed cell volume, and red blood cell count in the male workers exposed to EGME were significantly lower than in the controls. The frequency of anaemia in the exposed group (26.1%) was significantly higher than in the control group (3.2%). However, no differences were found between the female workers exposed and not exposed to EGME. After adjustment for sex, body mass index, and duration of employment, red blood cell count was significantly negatively associated with air concentrations of EGME, and haemoglobin, packed cell volume, and red blood cell count were significantly negatively associated with urinary concentrations of MAA. The pH of semen in the exposed workers was significantly lower than in the control workers, but there were no significant differences in the sperm count or sperm morphology between the exposed and control groups. CONCLUSION: It can be concluded that EGME is a haematological toxin, which leads to anaemia in the exposed workers. However, the data from this study did not support the theory of a spermatotoxic effect of EGME.

Twenty-eight-day repeated-dose inhalation exposure of rats to diethylene glycol monoethyl ether.

This study was carried out to provide information on the effects of inhalation of diethylene glycol monoethyl ether, a substance used in industry which may be accidentally inhaled by man. Sprague-Dawley CD rats were exposed by inhalation to a test atmosphere containing diethylene glycol monoethyl ether in a nose-only exposure system for 6 hr a day, 5 days a week for 28 days. Mean exposure levels were 0. 09, 0.27, and 1.1 mg/liter. At the two lowest exposure levels the test substance was present entirely as vapor, but at the highest exposure level the test atmosphere was approximately equally divided by mass into respirable droplets (aerosol) and vapor. A comprehensive battery of toxicological evaluations including food consumption, body weight, clinical signs, hematology, and biochemistry revealed no evidence of a systemic effect of exposure. Histopathological examination showed changes indicative of mild nonspecific irritation in the upper respiratory tract of rats exposed at the two highest exposure levels. These changes consisted of foci of necrosis in the ventral cartilage of the larynx of rats exposed at 0.27 or 1.1 mg/liter and an increase in eosinophilic inclusions in the olfactory epithelium of the nasal mucosa of rats exposed at 1.1 mg/liter. The no observed adverse effect level for systemic effects was 1.1 mg/liter and the no observed adverse effect level for signs indicative of mild nonspecific irritation of the upper respiratory tract was 0.09 mg/liter.

Serum ethylene glycol by high-performance liquid chromatography.

A high-performance liquid chromatography procedure for detection and quantitation of ethylene glycol in serum is described. Ethylene glycol and internal standard are derivatized with benzoyl chloride under alkaline conditions, purified by solid-phase extraction and analyzed by HPLC with UV detection. Analytical recovery of ethylene glycol ranges between 96 and 103%. The calibration curve is linear from 20 to 2000 mg/l. The limits of detection and quantitation are 10 and 20 mg/l, respectively. Assay imprecision is 4.8% or less. The assay is free from common interferences and provides increased sensitivity, improved precision and extended linearity.

Pharmacokinetics of ethylene glycol. I. Plasma disposition after single intravenous, peroral, or percutaneous doses in female Sprague-Dawley rats and CD-1 mice.

The pharmacokinetics of [1,2-14C]ethylene glycol (EG) were evaluated in female Sprague-Dawley rats and CD-1 mice to characterize the plasma disposition after intravenous (IV), peroral (PO), and percutaneous (PC) doses. Rats were given doses of 10 or 1000 mg/kg by each route, and additional PO doses of 400, 600, or 800 mg/kg. Mice were also given IV and PO (bolus gavage) doses of 10 or 1000 mg/kg, and additional PO doses of 100, 200, or 400 mg/kg. PC doses in mice were 100 or 1000 mg/kg, and both species were given a 1000 mg/kg PC dose with a 50% (w/w) aqueous solution (2 ml/kg) to simulate antifreeze exposure. Results from this study have shown that orally-administered EG is very rapidly and almost completely absorbed in both rats and mice, with a bioavailable fraction of 92-100% in rats and similar percentages at the higher doses in mice. In contrast, the absorption of cutaneously applied EG is comparatively slow in both species. A species difference in the overall absorption of PC doses was demonstrated, with higher recoveries of 14C observed after PC doses in mice than for rats and a greater penetration of 14C after applying a 50% aqueous PC dose in mice than in rats, as evidenced by quantifiable plasma 14C concentrations only in mice. The major metabolites in both rats and mice are CO2 and glycolate. Oxidative metabolic pathways are saturated at high PO doses in both species, resulting in a shift from exhaled CO2 as the major excretion route to urinary excretion. The capacity to metabolize more completely EG to CO2 at low doses seems to be greater in the mouse than in the rat, as evidenced by the absence of urinary oxalate from EG-dosed female mice, and saturation of metabolic pathways at a comparatively lower dose in mice than for rats. This evidence suggests that dose-dependent changes in EG excretion in female Sprague-Dawley rats and CD-1 mice probably resulted from capacity-limited effects on EG metabolic pathways for the production of CO2 and a compensatory urine clearance of glycolate. Results from the present study corroborate previous observations in rats for the lower doses, but demonstrate a substantial difference in single-dose pharmacokinetics for IV and PO 1000 mg/kg doses in mice vs. rats. In summary, these data indicate that mice show a nonlinear plasma disposition of total radioactivity (EG and its metabolites) as dose is increased, whereas in rats plasma kinetics were linear over the dose range evaluated, whereas excretion kinetic patterns were nonlinear in both species as dose is increased.

Pharmacokinetics of ethylene glycol. III. Plasma disposition and metabolic fate after single increasing intravenous, peroral, or percutaneous doses in the male Sprague-Dawley rat.

1. The pharmacokinetic fate of [1,2-14C]-ethylene glycol (EG) was evaluated in the male Sprague-Dawley rat in order to characterize its overall uptake and elimination. Doses of 10 and 1000 mg/kg were administered by the intravenous (i.v.), peroral (p.o.), or percutaneous (p.c.) route; additional doses of 400, 600 and 800 mg/kg were evaluated by the p.o route. 2. Baseline data obtained by the i.v. route for bioavailability comparisons showed that while plasma radioactivity concentrations declined in a biexponential manner with t1/2 beta of 26-37 h, the disappearance of unmetabolized EG from the plasma was quite rapid (t1/2 beta of 0.8-1.2 h). Peroral doses were rapidly and almost completely absorbed, showing t1/2 abs in the order of minutes, and a bioavailable fraction for unmetabolized EG of 92-100%. Conversely, EG applied to rat skin was slowly and rather poorly absorbed, showing t1/2 abs which were an order of magnitude longer than for comparable p.o. and i.v. doses, and a bioavailability of approximately 22%. 3. The major route of elimination for the 10 mg/kg dose by any route was by metabolism to 14CO2 and exhalation, while urinary elimination of 14C was the secondary excretion pathway. 4. Plasma clearance of 14C was linear with increases in p.o. doses over the 400-800 mg/kg range, with AUC proportional to dose for these and the 10 mg/kg p.o. dose levels. However, a dose-dependent shift in excretion routes was observed following the p.o. 1000 mg/kg dose, with urine becoming the major excretion route, and similar capacity limited pharmacokinetics were observed for the i.v. 1000 mg/kg dose. Plasma pharmacokinetic data for unchanged EG after i.v. and p.o. doses demonstrated an apparent first-order kinetic behaviour between the 10 and 1000 mg/kg dose levels for the disappearance of EG. 5. Following both i.v. and p.o doses, dose-independent relationships were seen in the values obtained for the area under the plasma curve (AUC infinity), the total clearance of EG (CltotalEG), mean residence time (MRT infinity), apparent volume of distribution at steady state (Vdss), the terminal half-life (t1/2 beta) and the renal and metabolic clearance values. However, this dose-linear plasma time course was not apparent from the dose-dependent excretion profiles for these two exposure routes. 6. Increases in urinary 14C-glycolate were also observed when the i.v. or p.o. doses were increased from 10 to 1000 mg EG/kg, indicating that metabolism of EG makes a substantial contribution to AUC infinity in the beta disposition phase of the plasma curves for this high dose. Oxalate, a metabolite found in man after EG exposure, was detected at very low levels after both the 10 and 1000 mg/kg dose levels and by either i.v or p.o. routes. 7. Thus, EG given by three different routes demonstrated apparent first-order pharmacokinetic behaviour for disposition in and the elimination from plasma in the male rat, but dose-dependent changes occurred for the elimination of metabolites in urine and as 14CO2 after single i.v. and p.o. doses, but not for the p.c. routes.

Utility of the serum osmol gap in the diagnosis of methanol or ethylene glycol ingestion.

Ingestion of methanol or ethylene glycol is a toxicologic emergency. The osmolal gap has been widely advocated as a screen for serum methanol or ethylene glycol. Unfortunately, for several reasons the osmolal gap fails in this capacity. First, an accurate serum osmolality can often not be obtained. Second, the calculated serum osmolarity will vary greatly, depending on the formula used to estimate it. Third, ethylene glycol has such a large molecular weight that even toxic amounts may contribute minimally to a patient's overall osmolality. Finally, because of metabolism, little ethylene glycol or methanol may be present when a patient presents with toxicity. These limitations invalidate the osmolal gap as a screen for ethylene glycol or methanol ingestion.

Simultaneous determination of ethylene glycol and its major toxic metabolite, glycolic acid, in serum by gas chromatography.

We developed a gas-chromatographic procedure for the simultaneous determination of ethylene glycol (EG) and its major toxic metabolite, glycolic acid (GA), suitable for clinical use in instances of EG intoxication. After serum protein precipitation with acetonitrile (containing internal standard), the supernate is treated with 2,2-dimethoxypropane (containing dimethylformamide) to remove water, and the volume is then reduced by evaporation to <100 microL of dimethylformamide (but not to dryness). After trimethylsilyl derivatization, the resulting derivatives are analyzed by capillary column gas chromatography. Only 100 microL of serum is required and the entire determination, including calibrators and controls, takes <2 h. The method gives a linear response to at least 10 g/L EG and 5 g/L GA and has a limit of detection <10 mg/L. Intraassay CV is < or = 2.8% for EG (100 and 1000 mg/L) and GA (100 and 500 mg/L); between-day CV is < or = 6.5%. The absolute recovery from serum was 91% for EG and 77-82% for GA (200 and 2000 mg/L each). Relative to calibrators prepared bovine serum albumin (70 g/L), the recovery was 99-104% for EG (100 - 5000 mg/L) and 95-105% for GA (50 - 2500 mg/L). No clinically important interference was detected for >60 exogenous or endogenous compounds and drugs.

Ethylene glycol acute poisoning treatment results in Kraków in the years 1990-1994.

An analysis of ethylene glycol acute intoxication treatment results was performed in a group of 36 patients hospitalized within a five year period. Mean serum and urine glycol concentrations in the analyzed population ranged from 0-851 mg/dl (mean = 130 mg/dl) and from 12.4 to 930.0 mg/dl (mean 333 mg/dl), respectively. At the time of admission to the clinic 15 of 36 patients were deeply unconscious and mean acid-base balance values were as follows: pH 6.99, pCO2 16.7 mmHg, pO2 140.1 mmHg, HCO3 6.36 mmol/l, BE -29.6 mmol/l. Because of respiratory failure 21/36 patients (58.3%) required controlled ventilation and 24/36 (66.7%) underwent dialysis. Sixteen patients (44.4%) developed acute renal failure. Mean hospitalization period was 16 days (1-53). Eighteen patients (50%) died. The direct death mechanism in 15 patients (83.3%) was asystolia and in the remaining individuals other circulatory disturbances. The main reasons of high mortality rate were multiple organ damages secondary to severe metabolic acidosis.

4-Methylpyrazole and hemodialysis in ethylene glycol poisoning.

CASE REPORTS: Two patients severely intoxicated with ethylene glycol became anuric and were treated by hemodialysis and the antidote, 4-methylpyrazole. On admission, their plasma ethylene glycol concentrations were 0.42 and 3 g/L respectively and no alcohol was detected. The elimination of 4-methylpyrazole in the dialysate represented 45% of the total body elimination. Clearances of 4-methylpyrazole by hemodialysis were 80 mL/min and 52 mL/min respectively. RESULTS: In such cases, the authors propose infusion of a 4-methylpyrazole loading dose of 10-20 mg/kg before dialysis and intravenous infusion of 1-1.5 mg/kg/h during the 8-12 hours of hemodialysis to compensate the loss in dialysate.

A novel derivatization of ethylene glycol from human serum using 4-carbethoxyhexafluorobutyryl chloride for unambiguous gas chromatography-chemical ionization mass spectrometric identification and quantification.

Ethylene glycol poisoning is a common problem and identification of ethylene glycol in serum is important for both medical and legal purposes. Other glycols like propylene glycol, 2,3-butanediol, and diethylene glycols are also abused. Common derivatization techniques to form cyclic ester of ethylene glycol work only for 1,2-diols and cannot derivatize other glycols like diethylene glycol. The authors describe novel derivatization technique of ethylene glycol (Molecular weight 62) using 4-carbethoxyhexafluorobutyryl chloride that produces a distinct protonated molecular ion peak at m/z 563 in chemical ionization mode, thus aiding in unambiguous confirmation of ethylene glycol. The authors' novel derivatization technique is not limited to 1,2-diols and can derivatize any glycol. It yields less volatile derivatives than any conventional derivatives and is free from interference from the serum matrix. Moreover, those glycols produce distinctively different mass spectra compared to ethylene glycol. Quantitation of ethylene glycol can be easily achieved by using 1,2-butanediol as an internal standard. The assay showed a within run and between run precision of 6.7% and 8.2% and a linearity from 1.1-36.1 mmol/L (70-2,240 micrograms/mliters) of serum ethylene glycol concentration.

A report of accidental ethylene glycol ingestion in 2 siblings.

We describe a case of 2 siblings aged 2 1/2 and 3 1/2 yrs accidentally poisoned by ethylene glycol ingestion. We found estimating the level of ethylene glycol in plasma by calculation of osmolar gap too insensitive to be of value and advocate the availability of a specific method. In our study only one of the 2 children had a toxic level of ethylene glycol but assay by conventional assay and by proton magnetic resonance spectroscopy (1HMRS) of toxic metabolites viz glycolate, glyoxylate and oxalate showed both to be excreting grossly elevated levels. This indicates the desirability of assaying the toxic metabolites of the glycol as well as the parent compound in assessing ingestions.

Gas chromatographic-mass spectrometric identification and quantitation of ethylene glycol in serum after derivatization with perfluorooctanoyl chloride: a novel derivative.

Ethylene glycol poisoning is a common clinical problem and identification as well as quantitation of ethylene glycol in serum is important for medical and legal purposes. Most investigators described determination of ethylene glycol by gas chromatography without derivatization or derivatives forming a molecular ion < 200. We describe a novel derivatization technique of ethylene glycol using perfluorooctanoyl chloride, after extraction from serum using acetone. This derivative has a molecular mass of 854 and produces a base peak at m/z 441 and other diagnostic strong peaks for unambiguous identification. Moreover, this derivative is less volatile and is free from interferences from endogenous serum components. Quantitation can be achieved by using 1,4-butanediol as an internal standard. The assay showed within-run and between-run precision of 7.2% and 8.0%, respectively, and linearity over the serum ethylene glycol concentration range 70-2240 micrograms/ml with a detection limit of 5 micrograms/ml.

Determination of 2-butoxyethanol and butoxyacetic acid in rat and human blood by gas chromatography-mass spectrometry.

A sensitive and selective gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed to simultaneously quantitate 2-butoxyethanol (BE) and butoxyacetic acid (BAA) in rat and human blood at low ng/g levels as pentafluorobenzoyl and pentafluorobenzyl derivatives, respectively. Analysis of 13C-labeled analogs of BE and BAA were found to improve the limits of quantitation to below 2 ng/g. Deuterium-labeled BE and BAA were used as internal standards. Calibration curves were generally linear over three orders of magnitude, with limits of quantitation of 16-18 ng/g for both BE and BAA, and 1.5 and 0.4 ng/g for [13C2]BE and [13C2]BAA, respectively, in human blood. Linearity in rat blood was similar, with limits of quantitation of 22 ng/g for BE and 5 ng/g for BAA. This method was developed for the support of mammalian metabolism studies and human biomonitoring studies involving exposure to BE or [13C2]BE.