The urinary metabolic profile of diethylene glycol methyl ether and triethylene glycol methyl ether in Sprague-Dawley rats and the role of the metabolite methoxyacetic acid in their toxicity.

Ethylene glycol ethers are a well-known series of solvents and hydraulic fluids derived from the reaction of ethylene oxide and monoalcohols. Use of methanol as the alcohol results in a series of mono, di and triethylene glycol methyl ethers. The first in the series, monoethylene glycol methyl ether (EGME or 2-methoxyethanol) is well characterised and metabolises in vivo to methoxyacetic acid (MAA), a known reproductive toxicant. Metabolism data is not available for the di and triethylene glycol ethers (DEGME and TEGME respectively). This study evaluated the metabolism of these two substances in male rats following single oral gavage doses of 500, 1000 and 2000 mg/kg for DEGME and 1000 mg/kg for TEGME. As for EGME, the dominant metabolite of each was the acid metabolite derived by oxidation of the terminal hydroxyl group. Elimination of these metabolites was rapid, with half-lives <4 h for each one. Both substances were also found to produce small amounts of MAA (~0.5% for TEGME and ~1.1% for DEGME at doses of 1000 mg/kg) through cleavage of the ether groups in the molecules. These small amounts of MAA produced can explain the effects seen at high doses in reproductive studies using DEGME and TEGME.

Neurotoxic effects of nephrotoxic compound diethylene glycol.

CONTEXT: Diethylene glycol (DEG) is an organic compound found in household products but also as an adulterant in medicines by acting as a counterfeit solvent. DEG poisonings have been characterized predominately by acute kidney injury (AKI), but also by delayed neurological sequelae such as decreased reflexes or face and limb weakness. OBJECTIVES: Characterizing the neurological symptoms of DEG poisoning in a subacute animal model would create a clearer picture of overall toxicity and possibly make mechanistic connections between kidney injury and neuropathy. METHODS: Male Wistar-Han rats were orally administered doses of 4 - 6 g/kg DEG every 12 or 24 h and monitored for 7 days. Urine was collected every 12 h and endpoint blood and cerebrospinal fluid (CSF) were collected for a renal plasma panel and total protein estimation, respectively. Motor function tests were conducted before and after treatment. Kidney and brain tissue was harvested for metabolic analysis. RESULTS: Of the 43 animals treated with DEG, 11 developed AKI as confirmed by increased BUN and creatinine levels. Renal and brain DGA accumulation was markedly increased in animals that developed AKI compared to animals without AKI. The total protein content in CSF in animals with kidney injury was markedly elevated compared to control and to treated animals without AKI. Significant decreases in forelimb grip strength and decreases in locomotor and rearing activity were observed in animals with AKI compared to control and to animals without AKI. DISCUSSION: Repeated dosing with DEG in an animal model produced nephrotoxic effects like those in studies with acute DEG administration. The decrease in motor function and increase in CSF protein were only present in animals that developed AKI. CONCLUSIONS: These studies show development of neurotoxicity in this DEG animal model and suggest that neurological symptoms are observed only when DGA accumulation and kidney injury also occur.

Catching Fakes: New Markers of Urine Sample Validity and Invalidity.

Urine drug testing is common for workplace drug testing, prescription management, emergency medicine and the criminal justice system. Unsurprisingly, with the significant consequences based upon the results of urine drug testing, a donor in need of concealing the contents of their sample is highly motivated to cheat the process. Procedures and safeguards ensuring sample validity are well known, and include measuring sample temperature at the time of collection, and laboratory measurements of creatinine, specific gravity and pH. Synthetic urine samples are available and are designed to deceive all aspects of urine drug testing, including validity testing. These samples are sophisticated enough to contain biological levels of creatinine, and are at a physiological pH and specific gravity. The goal of our research was to develop new procedures designed to distinguish authentic samples from masquerading synthetic samples. We aimed to identify substances in commercial synthetic urines not expected to be present in a biological sample distinguishing fake specimens. Additionally, we aimed to identify and employ endogenous compounds in addition to creatinine for identifying biological samples. We successfully identified two compounds present in synthetic urines that are not present in biological samples and use them as markers of invalidity. Four new endogenous markers for validity were successfully evaluated. Validity assessment was further aided by monitoring metabolites of nicotine and caffeine. When the method was applied to patient samples, 2% of samples were identified as inconsistent with natural urine samples, even though they met the current acceptance criteria for creatinine, pH and specific gravity.

Biomonitoring of 2-(2-alkoxyethoxy)ethanols by analysing urinary 2-(2-alkoxyethoxy)acetic acids.

2-Methoxyacetic and 2-ethoxyacetic acids are well known toxic metabolites of 2-alkoxyethanols. The use of 2-alkoxyethanols is now restricted, and the regulations have forced manufacturers to find substitutive solvents, 2-(2-alkoxyethoxy)ethanols. 2-(2-Alkoxyethoxy)ethanols resemble 2-alkoxyethanols, and their most hazardous similarity is their ability to metabolize to the 2-(2-alkoxyethoxy)acetic acids. In the present study, floor lacquerers' (n = 22) inhalation and total exposure to 2-(2-alkoxy)ethoxyethanols was measured. The measurements of inhalation exposure were done with charcoal tubes, and total exposure was biomonitored by urinalysis of 2-(2-alkoxyethoxy)acetic acids. The 8h inhalation exposures of floor lacquerers to 2-(2-methoxyethoxy)ethanol (DEGME), 2-(2-ethoxyethoxy)ethanol (DEGEE) and 2-(2-butoxyethoxy)ethanol (DEGBE) were in average 0.23 +/- 0.07 ppm (average+/-S.D., n = 3), 0.08 +/- 0.07 ppm (n = 16), and 0.05 +/- 0.03 ppm (n = 16), respectively. The excretions of 2-(2-methoxyethoxy)acetic acid (MEAA), 2-(2-ethoxyethoxy)acetic acid (EEAA) and 2-(2-butoxyethoxy)acetic acid (BEAA) were in average 4.9 +/- 4.3 mmol/mol creatinine, 9.3 +/- 8.0 mmol/mol creatinine and 9.2 +/- 7.4 mmol/mol creatinine, respectively. A linear relationship was found between the urinary 2-(2-alkoxyethoxy)acetic acid concentrations and the preceding 8-h occupational exposure to 2-(2-alkoxyethoxy)ethanol.

The effects of imipramine and iprindole on the metabolism of octopamine in the rat.

Acute injections of imipramine and iprindole in rats produced significant decreases in the concentration of p-hydroxyphenylglycol (pHPG), a neutral metabolite of octopamine in brain at 6 and 24 hr after the administration of drugs. The 24-hr urinary levels of both free and total pHPG were reduced to 25-29% of control with acute administration of imipramine, while iprindole produced a 30% decrease in free pHPG. With chronic administration of imipramine, concentrations of pHPG in brain returned to normal, while the 24-hr urinary levels were still decreased (to 24%). Octopamine in brain was unaltered after both single and repeated injections of imipramine. Thus, these data suggest that the turnover of octopamine in brain is reduced after acute administration of imipramine and iprindole, while after chronic treatment with imipramine, turnover of octopamine in brain has returned to control levels.

Analyses of ethylene glycol monoalkyl ethers and their proposed metabolites in blood and urine.

Glycol ethers are known to produce embryotoxic and teratogenic effects in a variety of animal species. In addition, testicular edema and tubular atrophy have been reported. The health effects of this class of compounds are not known in humans, despite the fact that these solvents are widely used in industry. In order to evaluate potential effects in humans, it is first necessary to estimate exposure in the workplace (environmental monitoring). However, in the case of glycol ethers traditional air monitoring may be ineffective because of the low volatility of these solvents and the possible significant exposure via the skin. Biological monitoring can be used to estimate glycol ether uptake by all routes of exposure. The compounds can be measured in blood or their metabolites quantitated in urine. These procedures are suggested for measuring 2-methoxyethanol, 2-ethoxyethanol and 2-butoxyethanol in blood. In addition, tentative procedures have been developed to measure the oxidized acidic metabolites, methoxyacetic acid and ethoxyacetic acid in urine as possible indices of exposure. All procedures have detection limits of less than 11 parts per million. These procedures are ready to be validated in workers exposed to these solvents.

Metabolism and excretion of 2-ethoxyethanol in the adult male rat.

The routes of 14C excretion following the administration of a single oral 230 mg/kg body weight dose of 2-ethoxyethanol [ethanol-1,2-14C] or 2-ethoxyethanol [ethoxy-1-14C] to male Sprague-Dawley rats were investigated. Elimination of the 14C by the urinary route accounted for 76 to 80% of the dose within 96 hr. The main pathway of biotransformation is oxidation to the corresponding acid, with some subsequent conjugation of the acid metabolite with glycine. The major metabolites, ethoxyacetic acid and N-ethoxy-acetyl glycine, representing 73 to 76% of the administered dose, were eliminated in the urine. The major difference in the metabolic profiles of the two radiochemicals was in the rate and amount of 14CO2 expired via the lung. Of the administered 14C, 11.7% of the ethoxy-labeled and 4.6% of the ethanol-labeled compounds were eliminated as CO2. The biological half-time was 9.9 +/- 1.5 hr for the ethoxy-labeled compound and 12.5 +/- 1.9 hr for the ethanol label. After administration of the ethanol-labeled compound, the only radiolabeled component found in the rat testes was identified as ethoxyacetic acid. Results of this study suggest that the reported testicular effects in the rat may be a result of tissue levels of ethoxyacetic acid.

Identification and quantitation of phenylethylene glycol in human and rat urine, and its elevation in phenylketonuria.

Phenylethylene glycol has been identified in rat and human urine using gas chromatography/chemical ionization/mass spectrometry. A method was developed for the quantitative analysis in urine of this phenylalanine metabolite and of p-hydroxyphenylethanol, a metabolite of tyrosine, by converting them to the pentafluoropropionyl derivatives and measuring them by selected ion monitoring. In human urine, about 90% of the phenylethylene glycol was present in a conjugated form (releasable by glusulase), but the reverse was true for rat urine, with about 90% being present in the unconjugated form. The excretion of free phenylethylene glycol (expressed as ng/mg creatinine) was 2.7-fold higher in a group of untreated phenylketonuric patients than in the control group, but the phenylketonuric patients excreted abnormally low amounts of 3-methoxy-4-hydroxyphenylethylene glycol. Intraperitoneal injections of L-phenylalanine in rats resulted in a small increase in the excretion of phenylethylene glycol. On the other hand, the injection of phenylethanolamine resulted in an 82-fold increase in the excretion of phenylethylene glycol, but phenylethylamine had no effect. These results indicate that the conversion of phenylethylamine to phenylethanolamine is the rate limiting step in this metabolic pathway.

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.

Measurement of ethylene glycol (ethane-1,2-diol) in biological specimens using derivatisation and gas-liquid chromatography with flame ionisation detection.

Ethylene glycol in plasma, urine or dialysis fluid is analysed as the phenylboronate derivative by mixing with acetonitrile/acidified 2,2-dimethoxypropane containing phenylboronic acid. After centrifugation, a portion of the supernatant is analysed directly by gas-liquid chromatography using a 3% OV-101 column at 150 degrees C and flame-ionisation detection. Propane-1,3-diol is used as a reactive internal standard. The limit of accurate measurement is at least 0.1 g/L and the linear range extends up to 5.0 g/L. No sources of interference have been identified.

Some aspects of calcium metabolism in a fatal case of ethylene glycol poisoning.

Laboratory results are presented for a patient who died following ingestion of an antifreeze solution containing ethylene glycol. It was observed that the measurement of osmolality, which is of value in the early stages of ethylene glycol poisoning, may give normal results if there are many hours delay between ingestion and admission. The hypocalcaemia which frequently accompanies ethylene glycol poisoning is shown to develop over several hours.

DNA single strand breaks induced by low levels of occupational exposure to styrene: the gap between standards and reality.

Styrene is a known mutagen and suspected carcinogen, used in the reinforced plastic industry. This study aims to identify the occurrence of DNA single strand breaks (SSBs) in workers exposed to styrene levels far below the recommended standards. We compared 26 exposed workers with 26 control subjects and found a significant increase in the incidence of DNA-SSBs in the exposed individuals. The levels of the biological indices of exposure (urinary mandelic and phenyl glyoxylic acids) were less than 25% of the recommended limits. Reduction of the threshold limit values/time-weighted-average (TLV-TWA) applied is strongly recommended.

Urinary biochemistry in occupational exposure to glycol ethers.

Glycol ethers and glycol ether acetates are dehydrogenated to alkoxyacetic acid congeners which may serve as biological indicators of exposure. The ethereal bond may also be cut in an oxidation reaction catalyzed by the mixed function oxidase. In case of ethylene glycol, the eventual endproduct is oxalic acid. Urinary oxalic acid and alkoxyacetic acid excretion together was found to relate to the decrease of the succinate dehydrogenase activity (SDH) as an indicator of renal mitochondrial effects. The excretion of ammonia by exposed workers was doubled as compared to controls. The excretion of chloride was found to be smaller in the exposed than in controls. The excretion of calcium and glycosaminoglycans (GAG) among exposed workers were similar compared to controls.

A rapid gas chromatographic method for determination of ethylene glycol in serum and urine.

Intoxications with the antifreeze constituent ethylene glycol (EG) occur infrequently, but may be fatal if not recognized and treated promptly. The aim of the present work was to develop an analytical method for rapid diagnosis of EG poisoning and for monitoring EG removal by hemodialysis. EG was measured by gas chromatography upon direct injection of serum or urine samples (50 microL diluted in 200 microL of distilled water containing 2,3-butanediol as internal standard). A 2-m x 2-mm glass column with Chromosorb 101 (80/100 mesh) separates these glycols within four minutes at 200 degrees C, using nitrogen as the carrier gas. The glycols 1,2- and 1,3-propanediol were separated from EG and the internal standard. Acetone, methanol, and isopropanol did not interfere with the analysis. The limit of quantitation of EG was close to 0.5 mM. Because no derivatization, extraction, or concentration procedures were necessary, EG may be determined quantitatively within 30 min, allowing for monitoring of hemodialysis, which should be performed for 15 h in severe cases. The diagnosis of ethylene glycol intoxication in a late stage may be secured by analysis of urine collected on admission.

Determination of urinary metabolites of alkyl cellosolves by solid phase extraction and GC/FID.

Alkyl cellosolves include ethylene glycol monomethylether, ethylene glycol monoethylether, ethylene glycol monobuthylether. And their urine metabolites are methoxyacetic acid, ethoxyacetic acid and butoxyacetic acid. The current analytical method for urinary alkoxyacetic acid is liquid-liquid phase extraction. But the liquid-liquid phase extraction method needs a more complex pre-treatment process and has a low recovery rate. We determined the appropriate extraction solvent and its flow rate. We also evaluated the non-absorptive rate and recovery rate according to particle size. Finally we developed a convenient solid phase extraction method for the analysis of urine cellosolve metabolites. As a result, the recovery rates for methoxyacetic acid, ethoxyacetic acid and butoxyacetic acid were 100.4 +/- 1.6%, 100.2 +/- 1.8% and 100.7 +/- 10.0% respectively, when acetone was used as the extraction solution. The most appropriate flow rate was 0.1 ml/min. At a particle size of 140-200 mesh, non-absorption percentages for methoxyacetic acid, ethoxyacetic acid, butoxyacetic acid were 3.2 +/- 0.3%, 1.0 +/- 0.1% and 1.1 +/- 0.1%, and the recovery rates according to particle size were similar. Further evaluation of the recovery rate and non-absorptive rate according to the mini column shape, stationary phase and recovery rate with various extracting solutions is required.

Early clinicopathologic findings in dogs ingesting ethylene glycol.

Fifteen dogs were given 9.5 ml of ethylene glycol/kg of body weight, orally. Physical examination and clinical laboratory findings were evaluated at 1 and 3 hours after ingestion. Three of these dogs were also evaluated at 6, 9, 12, 24, 48, and 72 hours after ingestion. At 1 and 3 hours, the dogs were depressed, ataxic, and polydipsic with increased urine output and serum osmolality. Plasma bicarbonate and urine osmolality were decreased. The osmolal and anion gaps were increased at 1 and 3 hours, respectively. Calcium oxalate crystalluria was first observed at 6 hours. Diminished renal excretory function was not evident until 48 hours. Depression, ataxia, metabolic acidosis, polydipsia, and polyuria in the presence of serum hyperosmolality were early (1 and 3 hour) findings that indicated ethylene glycol intoxication in dogs.

[Quantitative analysis of urinary ethylene glycol in rats exposed to ethylene oxide].

A gas chromatographic method was used for determining ethylene glycol in urine. The analytical procedure is based on an azeotropic distillation and on esterification with n-butyl boronic acid. The linear calibration curve was obtained up to 500 micrograms/ml of ethylene glycol. The detection limit was estimated to be 10 micrograms/ml and relative standard deviation was 3.5% for 100 micrograms/ml of ethylene glycol. This method was applied to determine the urinary excretion of ethylene glycol in rats exposed to ethylene oxide at various concentrations (from 50 to 500 ppm). The excretion amounts of ethylene glycol were observed to be dependent on the concentration of ethylene oxide exposed.

[Analysis of urinary metabolites of rats exposed to ethylene oxide].

Urinary metabolites of rats exposed to ethylene oxide(EO) were analyzed by gas chromatography-mass spectrometry(GC/MS). Male Wister rats(220-240 g) were exposed to EO for 6 hours. The urine was collected during 18 hours after the exposure and extracted with ethyl acetate. For the water-soluble metabolites, ethanol was added to the urine centrifuged and the supernatant fraction was evaporated to dryness. The residue of the extract was methylated with diazomethane and trimethylsilylated with bis(trimethylsilyl)trifluoroacetamide. Ethylene glycol, 2-hydroxymercapturic acid, 2-methylthioethanol and 2-mercaptoethanol were identified as the metabolites of EO. These results suggest that the inhaled EO was hydrolyzed to ethylene glycol, and conjugated with glutathion to form the mercapturic acid and methylthio metabolite.

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.

[The effects of nitroglycol at low concentrations (author's transl)].

In the first experiment of the present study, low concentrations of nitroglycol (ethylene glycol dinitrate) which are doses corresponding to the amounts of occupational exposure, were administered to 13 mongrel dogs and the changes of blood pressure (BP) cardiac output (CO), coronary blood flow (CBF) and femoral blood flow (FBF) were observed. A multichannel square wave electromagnetic blood flowmeter was used to measure the blood flows. After the administration of nitroglycol, fall of BP and increases of CO, CBF and FBF were observed. The increase of CBF were recognized more than 1 microgram of Ng per kg of body weight. As 50-200 micrograms/kg nitroglycol was administered intravenously, although a transient increase of CBF was found, the decrease of CBF for a relatively long period was followed. This fact suggested that a state of disadvantage for the coronary circulation was caused. In the second experiment, nitroglycol concentrations in blood and urine in 22 workers in a dynamite factory were measured by Götell's method. 0-145 ng/ml nitroglycol was detected in the blood after work, with high levels being noted in workers who had frequent exposure to skin absorption. The relationship between the nitroglycol concentration in blood of the workers and the experimental results in dogs was assessed and valuable suggestions concerning further research in the study of chronic exposure to nitroglycol at low concentration were obtained.