Deaths involving methylenedioxypyrovalerone (MDPV) in Upper East Tennessee.

Two deaths involving 3, 4-methylenedioxypyrovalerone (MDPV) are reported. MDPV is a synthetic cathinone stimulant found in "bath salts" with neurological and cardiovascular toxicity. Biological specimens were analyzed for MDPV by GC/MS and LC/MS. A White man was found dead with signs of nausea and vomiting after repeatedly abusing bath salts during a weekend binge. Femoral venous blood and urine had MDPV concentrations of 39 ng/mL and 760 ng/mL. The second fatality was a White man with a history of drug and bath salt abuse found dead at a scene in total disarray after exhibiting fits of anger and psychotic behavior. Femoral venous blood and urine had MDPV concentrations of 130 ng/mL and 3800 ng/mL. The blood and urine MDPV concentrations are within the reported recreational concentration ranges (blood 24-241 ng/mL and urine 34-3900 ng/mL). Both decedents' deaths were attributed to relevant natural causes in a setting of MDPV abuse.

Biotransformation of ethanol to ethyl glucuronide in a rat model after a single high oral dosage.

Ethyl glucuronide (EtG) is a minor ethanol metabolite that confirms the absorption and metabolism of ethanol after oral or dermal exposure. Human data suggest that maximum blood EtG (BEtG) concentrations are reached between 3.5 and 5.5h after ethanol administration. This study was undertaken to determine if the Sprague-Dawley (SD) rat biotransforms ethanol to EtG after a single high oral dose of ethanol. SD rats (male, n=6) were gavaged with a single ethanol dose (4 g/kg), and urine was collected for 3 h in metabolic cages, followed by euthanization and collection of heart blood. Blood and urine were analyzed for ethanol and EtG by gas chromatography and enzyme immunoassay. Blood and urine ethanol concentrations were 195±23 and 218±19 mg/dL, whereas BEtG and urine EtG (UEtG) concentrations were 1,363±98 ng equivalents/mL and 210±0.29 mg equivalents/dL (X ± standard error of the mean [S.E.M.]). Sixty-six male SD rats were gavaged ethanol (4 g/kg) and placed in metabolic cages to determine the extent and duration of ethanol to EtG biotransformation and urinary excretion. Blood and urine were collected up to 24 h after administration for ethanol and EtG analysis. Maximum blood ethanol, urine ethanol, and UEtG were reached within 4 h, whereas maximum BEtG was reached 6 h after administration. Maximum concentrations were blood ethanol, 213±20 mg/dL; urine ethanol, 308±34 mg/dL; BEtG, 2,683±145 ng equivalents/mL; UEtG, 1.2±0.06 mg equivalents/mL (X±S.E.M.). Areas under the concentration-time curve were blood ethanol, 1,578 h*mg/dL; urine ethanol, 3,096 h*mg/dL; BEtG, 18,284 h*ng equivalents/mL; and UEtG, 850 h*mg equivalents/dL. Blood ethanol and BEtG levels were reduced to below limits of detection (LODs) within 12 and 18 h after ethanol administration. Urine ethanols were below LOD at 18 h, but UEtG was still detectable at 24h after administration. Our data prove that the SD rat biotransforms ethanol to EtG and excretes both in the urine and suggest that it is similar to that of the human.

Morphine promotes apoptosis via TLR2, and this is negatively regulated by beta-arrestin 2.

We have previously reported that morphine induces apoptosis. However, the underlying molecular mechanisms remain to be elucidated. Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, modulates cell survival and cell death in various systems. Evidence indicates that beta-arrestin 2 acts as a negative regulator of innate immune activation by TLRs. Here, we investigated the roles of TLR2, the downstreaming mediator MyD88, and beta-arrestin 2 in morphine-induced apoptosis. We showed that overexpression of TLR2 in HEK293 cells caused a significant increase in apoptosis after morphine treatment. Inhibition of MyD88 by transfecting dominant negative MyD88 or overexpression of beta-arrestin 2 by transfecting beta-arrestin 2 full length plasmid in TLR2 overexpressing HEK293 cells attenuated morphine-induced apoptosis. Our study thus demonstrates that TLR2 signaling mediates the morphine-induced apoptosis, and beta-arrestin 2 is a negative regulator in morphine-induced, TLR2-mediated apoptosis.

Lidocaine iontophoresis mediates analgesia in lateral epicondylalgia treatment.

BACKGROUND AND PURPOSE: Iontophoresis transcutaneously delivers anti-inflammatory and analgesic drugs for the treatment of musculoskeletal dysfunction. Lidocaine is a local anaesthetic with analgesic but no anti-inflammatory properties. The purpose of this investigation was to examine the clinical use of lidocaine iontophoresis-mediated analgesia in a larger treatment algorithm for five patients with lateral humeral epicondylalgia. METHOD: The investigation was a case series design of five subjects, aged 52 (+/- 6) years, with epicondylalgia of 12-393 days' duration. At each treatment session, the patients received cryotherapy, cross-fibre massage and passive stretch. Between sessions analgesia was provided by an 80 mA-min low-current, long-duration lidocaine iontophoresis (LI) over a 24-hour period. Patients were treated on an every-other-day basis for a total of three treatment sessions. Clinical improvements were determined by triplicate measurements of dolorimetric force over the affected epicondyle prior to treatment 1 (baseline), prior to sessions 2 and 3, and one week following the last session. RESULTS: Patients demonstrated an increasing tolerance to dolorimetric force application prior to the next session. The force values prior to session 2 (3.1 (+/- 1.1) Newton (N)) and one week following the third session (3.4 (+/- 0.5) N) were significantly improved from the baseline values (2.1 (+/- 0.9) N). CONCLUSIONS: Pain associated with lateral epicondylalgia decreased, and function improved in all patients at the final measurement. One patient returned during the 90-day follow-up period to seek additional medical attention. This investigation documents the potential for analgesia provided by LI in the rehabilitation process of musculoskeletal dysfunction.

Papain: a novel urine adulterant.

The estimated number of employees in the United Stated screened annually for illicit drugs is approximately 20 million, with marijuana being the most frequently abused drug. Urine adulterants provide an opportunity for illicit drug users to obtain a false-negative result on commonly used primary drug screening methods such as the enzyme multiplied immunoassay technique and the fluorescence polarized immunoassay technique (FPIA). Typical chemical adulterants such as nitrites are easily detected or render the urine specimen invalid as defined in the proposed SAMHSA guidelines for specimen validity testing based on creatinine, specific gravity, and pH. Papain is a cysteine protease with intrinsic ester hydrolysis capability. The primary metabolite of the psychoactive chemical in marijuana, 11-norcarboxy-Delta9-tetrahydrocannibinol (THC-COOH), was assayed by FPIA in concentrations ranging from 25 to 500 ng/mL, at pH values ranging from 4.5 to 8, over the course of 3 days with papain concentrations ranging from 0 to 10 mg/mL. FPIA analysis of other frequently abused drugs: amphetamines, barbiturates, benzodiazepines, cocaine, opiates, and phencyclidine, along with gas chromatography-mass spectrometry (GC-MS) of THC-COOH and high-pressure liquid chromatography-ultraviolet detection (HPLC-UV) of nordiazepam was performed in order to determine if the mechanism of urine adulteration by papain was analyte specific. Control and adulterated urine specimens (n = 30) were assayed for creatinine, specific gravity, and pH to determine if papain rendered the specimens invalid based on the proposed SAMHSA guidelines. There was a direct pH, temperature, and time-dependent correlate between the increase in papain concentration and the decrease in THC-COOH concentration from the untreated control groups (p < 0.01). The average 72-h THC-COOH concentration decrease at pH 6.2 with a papain concentration of 10 mg/mL was 50%. Papain did not significantly decrease the concentration of the other drugs analyzed with the exception of nordiazepam. GC-MS of THC-COOH and HPLC-UV of nordiazepam revealed a 66% and 24% decrease in concentration of the respective analyte with 10 mg/mL papain after 24 h at room temperature (approximately 23 degrees C). No adulterated specimens were rendered invalid based on the SAMHSA guidelines. Immediate FPIA analysis is suggested to minimize the interfering effects of papain with regards to primary drug screening.

Sevoflurane analysis in serum by headspace gas chromatography with application to various biological matrices.

Sevoflurane is a nonflammable general anesthetic administered by inhalation of vaporized liquid that rapidly partitions out of aqueous biological matrices into a gaseous phase because of its volatility and hydrophobicity. We describe a headspace analysis of sevoflurane that can be performed without the use of an expensive automated headspace analyzer. Sevoflurane standards (0-109 mg/L) and quality control specimens (12.2 and 72.9 mg/L) were prepared and quantitated on an intraday and interday basis. Headspace gas was manually injected (150 degrees C) with a 2.5-mL gas-tight syringe into a Perkin-Elmer model 8500 gas chromatograph equipped with a 6-ft x 2-mm i.d. glass column (100 degrees C) containing 0.2% Carbowax 1500 on Carbopak C packing with a flame-ionization detector (200 degrees C), which allowed for elution of the internal standard, 1-propanol (1.56 min), and sevoflurane (2.92 min). Linear regression of the peak-area ratios of sevoflurane to 1-propanol (6.38 g/L), versus the sevoflurane concentrations yielded an average intraday correlation coefficient of 0.989 (S.D. = 0.003, n = 5) and mean quality control specimen values of 14.19 mg/L (C.V. = 5.1%, n = 5) and 66.72 mg/L (C.V. = 3.3%, n = 5). The average interday standard curve correlation coefficient was 0.987 (S.D. = 0.01, n = 5), and the mean quality control specimen values were 12.22 mg/L (C.V. = 13.7%, n = 5) and 74.27 mg/L (C.V. = 8.7%, n = 5). The chromatographic method described produced accurate and reproducible results with a simple on-column headspace gas injection. This method allows for quantitation of sevoflurane in various biological matrices by chromatographic separation of the headspace gas in a sealed specimen container.

The distribution of sevoflurane in a sevoflurane induced death.

The distribution of sevoflurane (fluoromethyl 2,2,2,-trifluoro-1-(trifluoromethyl) ethyl ether) in blood, urine, liver, kidney, vitreous humor, and tracheal aspirate is presented from a subject with a sevoflurane induced death. Sevoflurane is a nonflammable general anesthetic administered by inhalation of vaporized liquid. Although general inhalation anesthetics have the potential to be fatal if not properly administered, the incidence of abuse is minute in comparison to other illicit drugs. Currently, there are no citations in the literature defining the body distribution of sevoflurane in a sevoflurane induced death. The decedent was found lying in a bed with an oxygen mask containing a gauze pad secured to his face. Three empty bottles and one partially full bottle of Ultane (sevoflurane) were found with the body in addition to two pill boxes containing a variety of prescription and non-prescription drugs. Serum, urine and gastric contents from the deceased were screened for numerous drugs and metabolites using a combination of thin layer chromatographic, colorimetric and immunoassay techniques. Analysis of biological specimens from the deceased revealed the presence of: amphetamine, caffeine, pseudoephedrine, nicotine, nicotine metabolite, and valproic acid. Sevoflurane concentrations were determined by headspace gas chromatography with flame ionization detection and revealed concentrations of 26.2 microg/mL in the blood, 105 microg/mL in the urine, 31.9 microg/mL in the tracheal aspirate, 86.7 microg/mL in the vitreous humor, 30.8 mg/kg in the liver, and 12.8 mg/kg in the kidney. The decedent had pathologies consistent with respiratory suppression including pulmonary atelectasis, pulmonary edema, and neck vein distention. The official cause of death was respiratory suppression by sevoflurane and the manner of death was unclear.

Dose-response effects of chronic lithium regimens on spatial memory in the black molly fish.

Lithium is widely used in the management of bipolar disorder, yet memory impairment is a serious side effect. To assess the effects of lithium on spatial working and reference memories, we have employed a plus maze utilizing spontaneous alternation (SA) and place-learning paradigms in two experiments with the black molly fish. Four treatment groups were gavaged with 20 microl of a 10, 100, or 1000 mM lithium chloride (LiCl) solution or ddH(2)O vehicle every 12 h for 22 to 24 days. On Day 15, subjects began an 8-day SA task or a 10-day place-learning task. Results indicate that there is a significant difference in SA performance among the treatment groups for Days 1, 2, and 3. Results of the place-learning task indicate that the 1 M dose group needed significantly more trials to reach criterion and made significantly fewer correct first choices than the other dose groups. Capillary ion analysis determinations of plasma and brain lithium levels illustrate linear dose-response relationships to doses administered. Regression analyses indicate that there is a relationship between SA performance and plasma/brain lithium levels during the initial part of testing. Collectively, the results indicate that chronic lithium administration impairs spatial working and reference memories.

A fatal drug interaction between oxycodone and clonazepam.

A case is presented of a fatal drug interaction caused by ingestion of oxycodone (Oxycontin) and clonazepam (Klonapin). Oxycodone is an opium alkaloid used in long-term pain management therapy. Clonazepam is a benzodiazepine used for the treatment of seizures and panic disorders. The Drug Abuse Warning Network (DAWN) has reported an increase of 108% in the last two years of emergency department episodes related to Oxycontin. Six billion prescriptions were written for Oxycontin in the year 2000, an 18-fold increase from four years previous (1). Oxycontin has recently gained enormous notoriety at the local and national levels; however, there are very few previously documented cases of lethal drug interactions between oxycodone and clonazepam. Synergistic effects between these two drugs are postulated to arise from different agonistic mechanisms producing similar physiological changes. It is also theorized that clonazepam may inhibit the metabolism of oxycodone. A 38-year-old white female was found dead in Jefferson County, Tennessee in March of 2001. The deceased had physical evidence of previous drug abuse and positive serological findings of hepatitis B and C. Prescription pill bottles filled under the name of the deceased, as well as another name, were found with the body. Serum, urine and gastric contents from the deceased were screened for numerous drugs and metabolites using a combination of thin layer chromatography and immunoassay techniques (EMIT and FPIA). Analysis of biological specimens from the deceased revealed the presence of: benzodiazepines, opiates (oxycodone), and trazodone metabolites in the serum; cannabinoids, benzodiazepines, opiates (oxycodone), trazodone, trazodone metabolites, nicotine, and nicotine metabolite in the urine; and benzodiazepines, opiates (oxycodone), nicotine, and nicotine metabolite in the gastric contents. Quantitative analyses for clonazepam was performed by high performance liquid chromatography (HPLC) and revealed a plasma concentration of 1.41 microg/mL. Plasma oxycodone and urine 11-nor-carboxy-delta-9-tetrahydrocannabinol concentrations were determined by gas chromatography/mass spectrometry and revealed concentrations of 0.60 microg/mL and 27.9 ng/mL, respectively. The deceased had pathologies consistent with severe central nervous system (CNS) and respiratory depression produced by high concentrations of clonazepam and oxycodone including collapsed lungs, aspirated mucus, and heart failure. The pathologies were sufficient to cause death, which was officially attributed to a drug overdose; however, the manner of death was unknown.

Determination of chromate adulteration of human urine by automated colorimetric and capillary ion electrophoretic analyses.

Various chemicals can be added to urine specimens collected for drug analysis to abnormally elevate ionic concentrations and/or interfere with either immunoassay urine drug-screening procedures or gas chromatographic-mass spectrometric confirmation techniques. One such adulterant, "Urine Luck" (formula 5.3), has been identified in our previous research to contain potassium dichromate. Screening of suspected adulterated specimens and confirmation of the adulterant are important for forensic drug screening. The application and comparison of automated colorimetric and capillary ion electrophoretic techniques for the detection, confirmation, and quantitation of chromate adulteration of urine specimens were the purpose of this investigation. Thirty-six urine specimens suspected of adulteration were analyzed for chromate by colorimetric analysis with diphenylcarbazide. Duplicate aliquots were analyzed for chromate by capillary ion electrophoresis. Results of the colorimetric chromate analyses revealed a mean chromate concentration of 929 microg/mL with a standard error of 177 microg/mL and a range of 30 to 5634 microg/mL. Results of the capillary ion electrophoresis chromate analyses revealed a mean chromate concentration of 1009 microg/mL with a standard error of 218 microg/mL and a range of 20 to 7501 microg/mL. The correlation coefficient between the capillary ion electrophoretic and colorimetric chromate results was r = 0.9669. Application of the automated diphenylcarbazide colorimetric technique provides rapid determination of chromate adulteration of a urine specimen. Capillary ion electrophoresis offers a separation technique to confirm the presence of chromate in suspected adulterated specimens. The excellent correlation between these methods substantiates their application to forensic testing as screening and/or confirmation techniques.

Failure to detect dexamethasone phosphate in the local venous blood postcathodic lontophoresis in humans.

STUDY DESIGN: A single-blind, 2-factor (4 treatments by 8 time points) repeated-measures study design. OBJECTIVE: To analytically determine dexamethasone and dexamethasone phosphate concentrations in plasma derived from proximal effluent venous blood, following cathodic iontophoresis. METHODS AND MEASURES: Six volunteers received the following dexamethasone phosphate (2.5 ml, 4 mg/ml) treatments to their wrists on separate occasions: cathodic iontophoresis (4 mA, 10 minutes or 4 mA, 20 minutes), passive application (10 or 20 minutes). Plasma samples from the ipsilateral antecubital vein were obtained 10 minutes prior to and half way through the treatment (5 or 10 minutes), at the end of the treatment (10 or 20 minutes), and posttreatment (15, 30, 60, 90, and 120 minutes). The present investigation examined: (1) the sensitivity and linearity of extraction and analysis of dexamethasone and dexamethasone phosphate; (2) the necessity for determining both; and (3) the plasma levels from proximal effluent venous blood following cathodic iontophoresis. RESULTS: The aggregate (n = 18) of the 6-point standard curves were linear for dexamethasone (r > 0.974) and dexamethasone phosphate (r > 0.829). In vitro dephosphorylation of dexamethasone phosphate to dexamethasone occurred in plasma at 37 degrees C and during freeze-thaw. Measurable dexamethasone or dexamethasone phosphate concentrations were absent at all time points and under all conditions in the human subjects. CONCLUSIONS: These results demonstrate the sensitivity of the current assay and the need for evaluating both forms of the drug, as in vitro dephosphorylation results in the presence of dexamethasone and dexamethasone phosphate in samples. Absence of measurable dexamethasone or dexamethasone phosphate in the proximal effluent venous blood may require re-evaluation of the extent of drug delivery during the clinical iontophoresis of dexamethasone phosphate.