A modified gelatin zymography technique incorporating total protein normalization.

Gelatinase zymography is a commonly used laboratory procedure; however, variability in sample loading and concentration reduce the accuracy of quantitative results obtained from this technique. To facilitate normalization of gelatinase activity by loaded protein amount, we developed a protocol using the trihalocompound 2,2,2-trichloroethanol to allow for gelatin zymography and total protein labeling within the same gel. We showed that detected protein levels increased linearly with loading, and describe a loading concentration range over which normalized gelatinase activity was constant. We conclude that in-gel total protein detection is feasible in gelatin zymography and greatly improves comparison of gelatinase activity between samples.

In vivo effects of naproxen, salicylic acid, and valproic acid on the pharmacokinetics of trichloroethylene and metabolites in rats.

It was recently demonstrated that some drugs modulate in vitro metabolism of trichloroethylene (TCE) in humans and rats. The objective was to assess in vivo interactions between TCE and three drugs: naproxen (NA), valproic acid (VA), and salicylic acid (SA). Animals were exposed to TCE by inhalation (50 ppm for 6 h) and administered a bolus dose of drug by gavage, equivalent to 10-fold greater than the recommended daily dose. Samples of blood, urine, and collected tissues were analyzed by headspace gas chromatography coupled to an electron capture detector for TCE and metabolites (trichloroethanol [TCOH] and trichloroacetate [TCA]) levels. Coexposure to NA and TCE significantly increased (up to 50%) total and free TCOH (TCOHtotal and TCOHfree, respectively) in blood. This modulation may be explained by an inhibition of glucuronidation. VA significantly elevated TCE levels in blood (up to 50%) with a marked effect on TCOHtotal excretion in urine but not in blood. In contrast, SA produced an increase in TCOHtotal levels in blood at 30, 60, and 90 min and urine after coexposure. Data confirm in vitro observations that NA, VA, and SA affect in vivo TCE kinetics. Future efforts need to be directed to evaluate whether populations chronically medicated with the considered drugs display greater health risks related to TCE exposure.

Comparative analysis of the relationship between trichloroethylene metabolism and tissue-specific toxicity among inbred mouse strains: liver effects.

Trichloroethylene (TCE) is a widely used organic solvent. Although TCE is classified as carcinogenic to humans, substantial gaps remain in our understanding of interindividual variability in TCE metabolism and toxicity, especially in the liver. A hypothesis was tested that amounts of oxidative metabolites of TCE in mouse liver are associated with hepatic-specific toxicity. Oral dosing with TCE was conducted in subacute (600 mg/kg/d; 5 d; 7 inbred mouse strains) and subchronic (100 or 400 mg/kg/d; 1, 2, or 4 wk; 2 inbred mouse strains) designs. The quantitative relationship was evaluated between strain-, dose-, and time-dependent formation of TCE metabolites from cytochrome P-450-mediated oxidation (trichloroacetic acid [TCA], dichloroacetic acid [DCA], and trichloroethanol) and glutathione conjugation [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione] in serum and liver, and various hepatic toxicity phenotypes. In subacute study, interstrain variability in TCE metabolite amounts was observed in serum and liver. No marked induction of Cyp2e1 protein levels in liver was detected. Serum and hepatic levels of TCA and DCA were correlated with increased transcription of peroxisome proliferator-marker genes Cyp4a10 and Acox1 but not with degree of induction in hepatocellular proliferation. In subchronic study, serum and liver levels of oxidative metabolites gradually decreased over time despite continuous dosing. Hepatic protein levels of CYP2E1, ADH, and ALDH2 were unaffected by treatment with TCE. While the magnitude of induction of peroxisome proliferator-marker genes also declined, hepatocellular proliferation increased. This study offers a unique opportunity to provide a scientific data-driven rationale for some of the major assumptions in human health assessment of TCE.

Comparative analysis of the relationship between trichloroethylene metabolism and tissue-specific toxicity among inbred mouse strains: kidney effects.

Trichloroethylene (TCE) is a well-known environmental and occupational toxicant that is classified as carcinogenic to humans based on the epidemiological evidence of an association with higher risk of renal-cell carcinoma. A number of scientific issues critical for assessing human health risks from TCE remain unresolved, such as the amount of kidney-toxic glutathione conjugation metabolites formed, interspecies and interindividual differences, and the mode of action for kidney carcinogenicity. It was postulated that TCE renal metabolite levels are associated with kidney-specific toxicity. Oral dosing with TCE was conducted in subacute (600 mg/kg/d; 5 d; 7 inbred mouse strains) and subchronic (100 or 400 mg/kg/d; 1, 2, or 4 wk; 2 inbred mouse strains) designs. The quantitative relationship was evaluated between strain-, dose, and time-dependent formation of TCE metabolites from cytochrome P-450-mediated oxidation (trichloroacetic acid [TCA], dichloroacetic acid [DCA], and trichloroethanol) and glutathione conjugation [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione], and various kidney toxicity phenotypes. In subacute study, interstrain differences in renal TCE metabolite levels were observed. In addition, data showed that in several strains kidney-specific effects of TCE included induction of peroxisome proliferator-marker genes Cyp4a10 and Acox1, increased cell proliferation, and expression of KIM-1, a marker of tubular damage and regeneration. In subchronic study, peroxisome proliferator-marker gene induction and renal toxicity diminished while cell proliferative response was elevated in a dose-dependent manner in NZW/LacJ but not C57BL/6J mice. Overall, data demonstrated that renal TCE metabolite levels are associated with kidney-specific toxicity and that these effects are strain dependent.

Kinetics of chloral hydrate and its metabolites in male human volunteers.

Chloral hydrate (CH) is a short-lived intermediate in the metabolism of trichloroethylene (TRI). TRI, CH, and two common metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA) have been shown to be hepatocarcinogenic in mice. To better understand the pharmacokinetics of these metabolites of TRI in humans, eight male volunteers, aged 24-39, were administered single doses of 500 or 1,500 mg or a series of three doses of 500 mg given at 48 h intervals, in three separate experiments. Blood and urine were collected over a 7-day period and CH, DCA, TCA, free trichloroethanol (f-TCE), and total trichloroethanol (T-TCE=trichloroethanol and trichloroethanol-glucuronide [TCE-G]) were measured. DCA was detected in blood and urine only in trace quantities (<2 microM). TCA, on the other hand, had the highest plasma concentration and the largest AUC of any metabolite. The TCA elimination curve displayed an unusual concentration-time profile that contained three distinct compartments within the 7-day follow-up period. Previous work in rats has shown that the complex elimination curve for TCA results largely from the enterohepatic circulation of TCE-G and its subsequent conversion to TCA. As a result TCA had a very long residence time and this, in turn, led to a substantial enhancement of peak concentrations following the third dose in the multiple dose experiment. Approximately 59% of the AUC of plasma TCA following CH administration is produced via the enterohepatic circulation of TCE-G. The AUC for f-TCE was found to be positively correlated with serum bilirubin concentrations. This effect was greatest in one subject that was found to have serum bilirubin concentrations at the upper limit of the normal range in all three experiments. The AUC of f-TCE in the plasma of this individual was consistently about twice that of the other seven subjects. The kinetics of the other metabolites of CH was not significantly modified in this individual. These data indicate that individuals with a more impaired capacity for glucuronidation may be very sensitive to the central nervous system depressant effects of high doses of CH, which are commonly attributed to plasma levels of f-TCE.

Mechanistics of trichloroethylene mineralization by the white-rot fungus Trametes versicolor.

The white-rot fungus Trametes versicolor degraded trichloroethylene (TCE), a highly oxidized chloroethene, and produced 2,2,2-trichloroethanol and carbon dioxide as the main products of degradation, based on the results obtained using [13C]-TCE as the substrate. For a range of concentrations of TCE between 2 and 20 mg l(-1), 53% of the theoretical maximum chloride expected from complete degradation of TCE was observed. Laccase was shown to be induced by TCE, but did not appear to play a role in TCE degradation. Cytochrome P-450 appears to be involved in TCE degradation, as evidenced by marked inhibition of degradation of TCE in the presence of 1-aminobenzotriazole, a known inhibitor of cytochrome P-450. Our results suggested that chloral (trichloroacetaldehyde) was an intermediate of the TCE degradation pathway. The results indicate that the TCE degradation pathway in T. versicolor appears to be similar to that previously reported in mammals and is mechanistically quite different from bacterial TCE degradation.

An unusual trichloroethanol fatality attributed to sniffing trichloroethylene.

We report the death of a 28-year-old man due to sniffing a contact cement containing trichloroethylene. Initial testing revealed the presence of 80 mg/L trichloroethanol in cardiac blood, and the death was ruled as being due to trichloroethanol toxicity resulting from chloral hydrate ingestion. However, further investigation of the case revealed that the trichloroethanol resulted from trichloroethylene abuse. Subsequent targeted analysis for trichloroethylene, four months after the death, confirmed its presence in cardiac blood (1.1 mg/L), bile (4.5 mg/L), and liver (2.5 mg/kg). Trichloroethanol was initially detected during routine drug screening that employed gas chromatography (GC) using an HP-5 column with electron capture detection and subsequently quantitated by GC using the same column as for the initial screen, but with flame-ionization detection (FID); ethchlorvynol was the internal standard. Trichloroethylene was quantitated by headspace GC with a Restek Rtx-BAC1 column and FID; 1,1,1-trichloroethane was the internal standard.

Improved protein-crystal identification by using 2,2,2-trichloroethanol as a fluorescence enhancer.

The identification of initial lead conditions for successful protein crystallization is crucial for structural studies using X-ray crystallography. In order to reduce the number of false-negative conditions, an emerging number of fluorescence-based methods have been developed which allow more efficient identification of protein crystals and help to distinguish them from salt crystals. Detection of the native tryptophan fluorescence of protein crystals is one of the most widely used methods. However, this method can fail owing to the properties of the crystallized protein or the chemical composition of the crystallization trials. Here, a simple, fast and cost-efficient method employing 2,2,2-trichloroethanol (TCE) has been developed. It can be performed with a standard UV-light microscope and can be applied to cases in which detection of native tryptophan fluorescence fails. In four test cases this method had no effect on the diffraction properties of the crystals and no structural changes were observed. Further evidence is provided that TCE can be added to crystallization trials during their preparation, making this method compatible with high-throughput approaches.

Lack of formic acid production in rat hepatocytes and human renal proximal tubule cells exposed to chloral hydrate or trichloroacetic acid.

The industrial solvent trichloroethylene (TCE) and its major metabolites have been shown to cause formic aciduria in male rats. We have examined whether chloral hydrate (CH) and trichloroacetic acid (TCA), known metabolites of TCE, produce an increase in formic acid in vitro in cultures of rat hepatocytes or human renal proximal tubule cells (HRPTC). The metabolism and cytotoxicity of CH was also examined to establish that the cells were metabolically active and not compromised by toxicity. Rat hepatocytes and HRPTC were cultured in serum-free medium and then treated with 0.3-3mM CH for 3 days or 0.03-3mM CH for 10 days, respectively and formic acid production, metabolism to trichloroethanol (TCE-OH) and TCA and cytotoxicity determined. No increase in formic acid production in rat hepatocytes or HRPTC exposed to CH was observed over and above that due to chemical degradation, neither was formic acid production observed in rat hepatocytes exposed to TCA. HRPTC metabolized CH to TCE-OH and TCA with a 12-fold greater capacity to form TCE-OH versus TCA. Rat hepatocytes exhibited a 1.6-fold and three-fold greater capacity than HRPTC to form TCE-OH and TCA, respectively. CH and TCA were not cytotoxic to rat hepatocytes at concentrations up to 3mM/day for 3 days. With HRPTC, one sample showed no cytotoxicity to CH at concentrations up to 3mM/day for 10 days, while in another cytotoxicity was seen at 1mM/day for 3 days. In summary, increased formic acid production was not observed in rat hepatocytes or HRPTC exposed to TCE metabolites, suggesting that the in vivo response cannot be modelled in vitro. CH was toxic to HRPTC at millimolar concentrations/day over 10 days, while glutathione derived metabolites of TCE were toxic at micromolar concentrations/day over 10 days [Lock, E.A., Reed, C.J., 2006. Trichloroethylene: mechanisms of renal toxicity and renal cancer and relevance to risk assessment. Toxicol. Sci. 19, 313-331] supporting the view that glutathione derived metabolites are likely to be responsible for nephrotoxicity.

Application of cryopreserved human hepatocytes in trichloroethylene risk assessment: relative disposition of chloral hydrate to trichloroacetate and trichloroethanol.

BACKGROUND: Trichloroethylene (TCE) is a suspected human carcinogen and a common groundwater contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver by cytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) by alcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymorphisms are known to affect the elimination of ethanol. It is therefore possible that polymorphisms in CH metabolism will yield subpopulations with greater than expected TCA formation with associated enhanced risk of liver tumors after TCE exposure. METHODS: The present studies were undertaken to determine the feasibility of using commercially available, cryogenically preserved human hepatocytes to determine simultaneously the kinetics of CH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined. Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations. RESULTS: There was large interindividual variation in the Vmax values for both TCOH and TCA formation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent. Despite the large variation in Vmax values among individuals, disposition of CH into the two competing pathways was relatively constant. CONCLUSIONS: These data support the use of cryopreserved human hepatocytes as an experimental system to generate metabolic and genomic information for incorporation into TCE cancer risk assessment models. The data are discussed with regard to cellular factors, other than genotype, that may contribute to the observed variability in metabolism of CH in human liver.

Disposition and metabolism in 1-(2-chloroethyl)-3-(2',3',4'-tri-o-acetyl, ribopyranosyl)-1-nitrosourea in rats.

The antineoplastic activity in animals of 1-(2-chloroethyl)-3-(2',3',4'-tri-O-acetyl, ribopyranosyl)-1-nitrosourea (RPCNU) has been widely demonstrated. The present study deals with the disposition and the metabolism of three 14C-labeled species of RPCNU. The chemical plasma half-life of the drug was less than 5 min. Within the first min after injections, most of the radioactivity derived from ethyl-14C groups were recovered as volatile products. Among these, 2-chloroethanol was identified as a main component. Analysis of labeled species in urine after administration of [ethyl-14C]RPCNU showed that thiodiacetic acid and its sulfoxide were major metabolites of RPCNU (62% of the urinary radioactivity). Traces of N-acetylcarboxymethyl- and N-acetylhydroxyethylcysteine) were also detected. The only labeled species concentrating in particular tissues was that carrying the chloroethyl moiety. Uptake to high levels of [ethyl-14C]RPCNU did occur in liver, kidney, pancreas, thymus, and Harder's gland.

Aldehyde dismutation catalyzed by pulmonary carbonyl reductase: kinetic studies of chloral hydrate metabolism to trichloroacetic acid and trichloroethanol.

The kinetics of the NAD(P)(+)-linked aldehyde dismutation by pulmonary carbonyl reductase of guinea pig were studied using a highly hydrated substrate, chloral hydrate (CH). The enzyme irreversibly converted the substrate into trichloroacetic acid (TCA) and trichloroethanol (TCE) in the presence of the reduced or oxidized cofactors, of which NAD(P)+ gave a higher reaction rate than did NAD(P)H, and the concentration ratios of the two products (TCA plus TCE) to CH utilized were 1:1. In the NAD(P)(+)-linked reaction TCA was the predominant product and its amount was compatible with that of TCE plus NAD(P)H produced, whereas in the NAD(P)H-linked reaction equal amounts of TCA and TCE were formed and the cofactor was little oxidized. These results suggest that the enzyme oxidized the hydrated aldehydes to TCA with NAD(P)+ as the cofactor and reduced the unhydrated aldehyde to TCE with NAD(P)H. The steady-state kinetic measurements in the NADP(+)-linked CH oxidation were consistent with an ordered Bi Bi mechanism which is the same as that for the secondary alcohol oxidation by the enzyme. The dehydrogenase activity was inhibited competitively with respect to CH by a secondary alcohol substrate, propan-2-ol. The CH and propan-2-ol dehydrogenase activities were similarly inactivated by 2,4,6-trinitrobenzene-sulfonate, and NADP(H), several cofactor analogs and a cofactor-competitive inhibitor, Cibacron blue dye, protected against the inactivation, which suggest that lysine residues are essential for catalysis.

Trichloroethylene oxidative metabolism in plants: the trichloroethanol pathway.

Trichloroethylene (TCE) is a widespread and persistent environmental contaminant. Recently, plants, poplar trees in particular, have been investigated as a tool to remove TCE from soil and groundwater. The metabolism of TCE in plants is being investigated for two reasons: one, plant uptake and metabolism represent an important aspect of the environmental fate of the contaminant; two, metabolism pattern and metabolite identification will help assess the applicability of phytoremediation. It was previously shown that TCE metabolites in plants are similar to ones that result from cytochrome P450-mediated oxidation in mammals: trichloroethanol, trichloroacetate and dichloroacetate. Our measurements indicate that one of these metabolites, trichloroethanol, is further glycosylated in tobacco and poplar. The glycoside was detected in all tissues (roots, stems and leaves) in comparable levels, and was at least 10 fold more abundant than free trichloroethanol. The glycoside in tobacco was identified as the ss-D-glucoside of trichloroethanol by comparison of the mass spectra and the chromatographic retention time of its acetylation product to that of the synthesized standard. Trichloroethanol and its glucoside did not persist in plant tissue once plants are removed from TCE contaminated water, indicating further metabolism.

Binding of the general anesthetics chloroform and 2,2,2-trichloroethanol to the hydrophobic core of a four-alpha-helix bundle proteins.

The structural features of general anesthetic binding sites on proteins are being examined using a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work suggested that halothane binding to the four-alpha-helix bundle was improved by (1) introducing a cavity into the hydrophobic core and (2) substituting a methionine side-chain in place of an alpha-helical heptad e position leucine. In this study, the ability of the general anesthetics chloroform and 2,2,2-trichloroethanol to bind to the hydrophobic core of the four-alpha-helix bundle (Aalpha2-L38M)2 is explored. The halogenated alkane chloroform binds with a dissociation constant (Kd) = 1.4 +/- 0.2 mM, whereas 2,2,2-trichloroethanol binds with a Kd = 19.5 +/- 1.2 mM. The affinity of both general anesthetics for the hydrophobic core of the four-alpha-helix bundle approximates their whole animal effective concentration in 50% of test subjects' (EC50) values, as shown previously for halothane. Tryptophan phosphorescence decay rates at 77 K are accelerated by a factor of 4.5 by both bound halothane and chloroform, indicating that the heavy-atom effect is responsible for a portion of the observed fluorescence quenching. Because heavy-atom effects are operative only at short distances, the findings indicate that these general anesthetics are binding in the vicinity of the indole rings of W15 in the hydrophobic core of the four-alpha-helix bundle scaffold. The results indicate that chloroform, halothane and 2,2,2-trichloroethanol may occupy the same sites on protein targets.

The cholecystohepatic circulation of trichloroethylene and its metabolites in dogs.

In order to examine the cholecystohepatic circulation of trichloroethylene (TRI) and its metabolites, we injected the gallbladder with TRI and its metabolites, i.e. chloral hydrate (CH), free-trichloroethanol (F-TCE), trichloroacetic acid (TCA) and conjugated-trichloroethanol (Conj-TCE), using anesthetized dogs. The absorption rates of water from the gallbladder were 25-30% 2 h after administration for all substances. The absorption rates of substances were 65-70% in the CH, F-TCE and TRI groups, and 40-50% in the Conj-TCE and TCA groups 2 h after the administration. Conj-TCE in the blood absorbed from the gallbladder has a tendency to be directly transported to the venous system rather than to be taken into hepatocytes in the liver. All of the administered substances, in particular, F-TCE might be metabolized to other substances in the gallbladder.

Extrahepatic organs metabolism of inhaled trichloroethylene.

An extrahepatic circulation system for dogs was developed using a portal vein to right femoral vein bypass procedure. This system maintained nearly normal biochemical and physiological parameters, i.e. arterial blood pressure, heart rate, electrocardiogram, leukocyte and erythrocyte count, hematocrit, alkaline phosphatase, blood urea nitrogen, ammonia and creatinine, for 2 h. Thus, the system appears to be a valid technique for investigating extrahepatic metabolism. Dogs were exposed for 1 h to 500, 700 and 1500 ppm of trichloroethylene. Free-trichloroethanol, trichloroacetic acid and conjugated-trichloroethanol appeared in the blood and urine after 30 min of exposure. The amounts of metabolite formed by dogs with hepatic bypass were less than by similarly exposed dogs without hepatic bypasses, specifically 50-80%, 10% and 10-20% for free-trichloroethanol, trichloroacetic acid and conjugated-trichloroethanol, respectively. In addition, trichloroethylene exposure produced a smaller decrease in leukocyte counts in the hepatic bypass dogs than in the non-bypass dogs. This observation may indicate that the liver itself played some role in the elimination or increment of leukocyte counts in the blood.

Increased formic acid excretion and the development of kidney toxicity in rats following chronic dosing with trichloroethanol, a major metabolite of trichloroethylene.

The chronic toxicity of trichloroethanol, a major metabolite of trichloroethylene, has been assessed in male Fischer rats (60 per group) given trichloroethanol in drinking water at concentrations of 0, 0.5 and 1.0 g/l for 52 weeks. The rats excreted large amounts of formic acid in urine reaching a maximum after 12 weeks ( approximately 65 mg/24 h at 1 g/l) and thereafter declining to reach an apparent steady state at 40 weeks (15-20 mg/24 h). Urine from treated rats was more acidic throughout the study and urinary methylmalonic acid and plasma N-methyltetrahydrofolate concentrations were increased, indicating an acidosis, vitamin B12 deficiency and impaired folate metabolism, respectively. The rats treated with trichloroethanol developed kidney damage over the duration of the study which was characterised by increased urinary NAG activity, protein excretion (from 4 weeks), increased basophilia, protein accumulation and tubular damage (from 12 to 40 weeks), increased cell replication (at week 28) and evidence in some rats of focal proliferation of abnormal tubules at 52 weeks. It was concluded that trichloroethanol, the major metabolite of trichloroethylene, induced nephrotoxicity in rats as a result of formic acid excretion and acidosis.

Biliary excretion of trichloroethylene and its metabolites in dogs.

To examine the biliary excretion of trichloroethylene (TRI) and its metabolites, we carried out various experiments with TRI and its metabolites, i.e., chloral hydrate (CH), free-trichloroethanol (F-TCE) and trichloroacetic acid (TCA), using anesthetized dogs. The amount of biliary excretion was significantly increased with the administration of CH and F-TCE, whereas it remained at control levels with the administration of TRI and TCA. The substances excreted into bile were conducted in the form of conjugated-TCE (Conj-TCE) in over 90% of the CH, F-TCE and TRI administration groups. About 95% of these Conj-TCE were conjugated with glucuronic acid. The cumulative excretion ratios of substances and metabolites to dose were 20% for CH and F-TCE, and about 1% for TCA and TRI 2 h after administration.

Alteration of chloral hydrate metabolism in rats with carbon tetrachloride-induced liver damage.

The metabolism of chloral hydrate (CH) was investigated in the isolated perfused rat liver system. The experiments were performed on rats that were administered carbon tetrachloride (CCl4) subcutaneously for 15 weeks to induce chronic liver damage and on untreated rats. Clearance of CH from the perfusion system was lower in damaged liver than in control liver. In both groups, 50-70% of the added CH was excreted into perfusate as trichloroethanol (TCE) and trichloroacetic acid (TCA) within 120 min. The TCE/TCA ratio was 1:1.3 in the control group compared to 2:1 in the damaged liver group. The findings suggest that CH metabolism in the liver is affected by chronic damage.

Unusual death attributed to the combined effects of chloral hydrate, lidocaine, and nitrous oxide.

A case in which the death of a 2-year-old male child was the result of an acute intoxication with chloral hydrate, lidocaine, and nitrous oxide is presented. Trichloroethanol (TCE), the primary metabolite of chloral hydrate, was qualitatively detected by the Fujiwara reaction. Quantitation of TCE was carried out by gas chromatography-mass spectrometry (GC-MS) with the following results: plasma, 79.0 mg/L; urine, 31.0 mg/L; gastric contents, 454.0 mg/L; bile, 111.0 mg/L; vitreous, 40.2 mg/L; cerebrospinal fluid (CSF), 68.3 mg/L; and liver, 164 mg/kg. Lidocaine was quantitated by GC analysis using nitrogen-phosphorus detection with the following results: plasma, 11.9 mg/L; urine, 3.7 mg/L; gastric contents, 15.3 mg/L; bile, 19.0 mg/L; vitreous, 17.8 mg/L; CSF, 9.4 mg/L; and liver, 19.0 mg/kg. Nitrous oxide was quantitated in the blood with a value of 4.4 mL/L.