Biomedical subjects

Dennis J Crouch

Publications and source records attributed to Dennis J Crouch.

14 recordsLinked to original sources

PubMed2006

Recovery of drugs of abuse from the Immunalysis Quantisal oral fluid collection device.

Drug recovery from a new oral fluid collection device was assessed. The evaluation was performed in vitro at three physiologically relevant concentrations for the following substances: amphetamine, methamphetamine, morphine, codeine, cocaine, benzoylecgonine, methadone, oxazepam, and Delta(9)-tetrahydrocannabinol (THC). Drug-free and drug-fortified controls were prepared and their concentration verified by liquid chromatography-tandem mass spectrometry. Aliquots of the controls were then "collected" with the device (n=3) using the manufacturer's recommended procedure. Collected samples were stored for 12 h to simulate shipping before analysis. Fresh, non-"collected" aliquots of each pool (n=3) were concurrently analyzed. The drug recoveries from the Quantisal were expressed as a mean percentage of the concurrently analyzed aliquots that were not subjected to device collection. Recoveries for oxazepam exceeded 97%, for amphetamine and methamphetamine exceeded 93%, and for opioids all exceeded 91%. The recoveries of cocaine were >91% and >82% for its polar metabolite, benzoylecgonine. Especially noteworthy was the recovery of THC from the Quantisal collector (81.3-91.4%). When compared with recoveries reported from other collection devices, the Quantisal was clearly superior.

Chromatography, High Pressure Liquid↗

PubMed2006

A comparison of the cell phone driver and the drunk driver.

OBJECTIVE: The objective of this research was to determine the relative impairment associated with conversing on a cellular telephone while driving. BACKGROUND: Epidemiological evidence suggests that the relative risk of being in a traffic accident while using a cell phone is similar to the hazard associated with driving with a blood alcohol level at the legal limit. The purpose of this research was to provide a direct comparison of the driving performance of a cell phone driver and a drunk driver in a controlled laboratory setting. METHOD: We used a high-fidelity driving simulator to compare the performance of cell phone drivers with drivers who were intoxicated from ethanol (i.e., blood alcohol concentration at 0.08% weight/volume). RESULTS: When drivers were conversing on either a handheld or hands-free cell phone, their braking reactions were delayed and they were involved in more traffic accidents than when they were not conversing on a cell phone. By contrast, when drivers were intoxicated from ethanol they exhibited a more aggressive driving style, following closer to the vehicle immediately in front of them and applying more force while braking. CONCLUSION: When driving conditions and time on task were controlled for, the impairments associated with using a cell phone while driving can be as profound as those associated with driving while drunk. APPLICATION: This research may help to provide guidance for regulation addressing driver distraction caused by cell phone conversations.

Adult↗

PubMed2005

Oral fluid collection: the neglected variable in oral fluid testing.

The potential to use oral fluid as a drug-testing specimen has been the subject of considerable scientific interest. The ease with which specimens can be collected and the potential for oral fluid (OF) drug concentrations to reflect blood-drug concentrations make it a potentially valuable specimen in clinical as well as forensic settings. However, the possible effects of the OF collection process on drug detection and quantification has often been over looked. Several studies have documented that drug-contamination of the oral cavity may skew oral fluid/blood drug ratios and confound interpretation when drugs are smoked, insufflated or ingested orally. OF pH is predicted to have an effect on the concentration of drugs in OF. However, in a controlled clinical study, the effect of pH was less than that of collection technique. Mean codeine OF concentrations in specimens collected a non-stimulating control method were 3.6 times higher than those in OF collected after acidic stimulation. Mean codeine concentrations were 50% lower than control using mechanical stimulation and 77% of control using commercial collection devices. Several factors should be considered if a commercial OF collection device is used. In vitro collection experiments demonstrated that the mean collection volume varied between devices from 0.82 to 1.86 mL. The percentage of the collected volume that could be recovered from the device varied from 18% to 83%. In vitro experiments demonstrated considerable variation in the recovery of amphetamines (16-59%), opiates (33-50%), cocaine and benzoylecgonine (61-97%), carboxy-THC (0-53%) and PCP (9-56%). Less variation in collection volume, volume recovered and drug recovery was observed intra-device. The THC stability was evaluated in a common commercial collection protocol. Samples in the collection buffer were relatively stable for 6 weeks when stored frozen. However, stability was marginal under refrigerated conditions and poor at room temperature. Very little has been published on the efficacy of using IgG concentration, or any other endogenous marker, as a measure of OF specimen validity. Preliminary rinsing experiments with moderate (50 mL and 2 x 50 mL) volumes of water did not reduce the OF IgG concentration below proposed specimen validity criteria. In summary, obvious and more subtle variables in the OF collection may have pronounced effects on OF-drug concentrations. This has rarely been acknowledged in the literature, but should to be considered in OF drug testing, interpretation of OF-drug results and future research studies.

Drug Stability↗

PubMed2005

Age-dependent methamphetamine-induced alterations in vesicular monoamine transporter-2 function: implications for neurotoxicity.

Tens of thousands of adolescents and young adults have used illicit methamphetamine. This is of concern since its high-dose administration causes persistent dopaminergic deficits in adult animal models. The effects in adolescents are less studied. In adult rodents, toxic effects of methamphetamine may result partly from aberrant cytosolic dopamine accumulation and subsequent reactive oxygen species formation. The vesicular monoamine transporter-2 (VMAT-2) sequesters cytoplasmic dopamine into synaptic vesicles for storage and perhaps protection against dopamine-associated oxidative consequences. Accordingly, aberrant VMAT-2 function may contribute to the methamphetamine-induced persistent dopaminergic deficits. Hence, this study examined effects of methamphetamine on VMAT-2 in adolescent (postnatal day 40) and young adult (postnatal day 90) rats. Results revealed that high-dose methamphetamine treatment caused greater acute (within 1 h) decreases in vesicular dopamine uptake in postnatal day 90 versus 40 rats, as determined in a nonmembrane-associated subcellular fraction. Greater basal levels of VMAT-2 at postnatal day 90 versus 40 in this purified fraction seemed to contribute to the larger effect. Basal tissue dopamine content was also greater in postnatal day 90 versus 40 rats. In addition, postnatal day 90 rats were more susceptible to methamphetamine-induced persistent dopaminergic deficits as assessed by measuring VMAT-2 activity and dopamine content 7 days after treatment, even if drug doses were adjusted for age-related pharmacokinetic differences. Together, these data demonstrate dynamic changes in VMAT-2 susceptibility to methamphetamine as a function of development. Implications with regard to methamphetamine-induced dopaminergic deficits, as well as dopamine-associated neurodegenerative disorders such as Parkinson's disease, are discussed.

Age Factors↗

PubMed2004

Effects of vehicle on the uptake and elimination kinetics of capsaicinoids in human skin in vivo.

While the physiologic and molecular effects of capsaicinoids have been extensively studied in various model systems by a variety of administration routes, little is known about the uptake and elimination kinetic profiles in human skin following topical exposure. The present study evaluated the uptake and elimination kinetics of capsaicinoids in human stratum corneum following a single topical exposure to 3% solutions containing 55% capsaicin, 35% dihydrocapsaicin, and 10% other analogues prepared in three vehicles: mineral oil (MO), propylene glycol (PG), and isopropyl alcohol (IPA). Capsaicinoid solutions were evaluated simultaneously in a random application pattern on the volar forearms of 12 subjects using a small, single 150-microg dose. Capsaicin and dihydrocapsaicin were recovered from human skin using commercial adhesive discs to harvest stratum corneum from treated sites. Capsaicinoids were extracted from the stratum corneum-adhesive discs and quantified by liquid chromatography/mass spectroscopy (LC/MS). Both capsaicinoids were detected in stratum corneum 1 min after application with all vehicles and achieved a pseudo-steady state shortly thereafter. IPA delivered three times greater capsaicin and dihydrocapsaicin into the human stratum corneum than PG or MO at all time points investigated. The Cmax of capsaicin in IPA, PG, and MO was 16.1, 6.2, and 6.5 microg, respectively. The dihydrocapsaicin content was 60% of capsaicin with all vehicles. The estimated T(half) of capsaicin and dihydrocapsaicin in the three vehicles was similar (24 h). Thus, maximal cutaneous capsaicinoid concentrations were achieved quickly in the human stratum corneum and were concentration and vehicle dependent. In contrast, capsaicinoid half-life was long and vehicle independent.

2-Propanol↗

PubMed2003

Metabolism of capsaicin by cytochrome P450 produces novel dehydrogenated metabolites and decreases cytotoxicity to lung and liver cells.

Capsaicin is a common dietary constituent and a popular homeopathic treatment for chronic pain. Exposure to capsaicin has been shown to cause various dose-dependent acute physiological responses including the sensation of burning and pain, respiratory depression, and death. In this study, the P450-dependent metabolism of capsaicin by recombinant P450 enzymes and hepatic and lung microsomes from various species, including humans, was determined. A combination of LC/MS, LC/MS/MS, and LC/NMR was used to identify several metabolites of capsaicin that were generated by aromatic (M5 and M7) and alkyl hydroxylation (M2 and M3), O-demethylation (M6), N- (M9) and alkyl dehydrogenation (M1 and M4), and an additional ring oxygenation of M9 (M8). Dehydrogenation of capsaicin was a novel metabolic pathway and produced unique macrocyclic, diene, and imide metabolites. Metabolism of capsaicin by microsomes was inhibited by the nonselective P450 inhibitor 1-aminobenzotriazole (1-ABT). Metabolism was catalyzed by CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Addition of GSH (2 mM) to microsomal incubations stimulated the metabolism of capsaicin and trapped several reactive electrophilic intermediates as their GSH adducts. These results suggested that reactive intermediates, which inactivated certain P450 enzymes, were produced during catalytic turnover. Comparison of the rate and types of metabolites produced from capsaicin and its analogue, nonivamide, demonstrated similar pathways in the P450-dependent metabolism of these two capsaicinoids. However, production of the dehydrogenated (M4), macrocyclic (M1), and omega-1-hydroxylated (M3) metabolites was not observed for nonivamide. These differences may be reflective of the mechanism of formation of these metabolites of capsaicin. The role of metabolism in the cytotoxicity of capsaicin and nonivamide was also assessed in cultured lung and liver cells. Lung cells were markedly more sensitive to cytotoxicity by capsaicin and nonivamide. Cytotoxicity was enhanced 5 and 40% for both compounds by 1-ABT in BEAS-2B and HepG2, respectively. These data suggested that metabolism of capsaicinoids by P450 in cells represented a detoxification mechanism (in contrast to bioactivation).

Animals↗

PubMed2003

An evaluation of rapid point-of-collection oral fluid drug-testing devices.

New technology is currently being marketed to rapidly test oral fluids for drugs of abuse at the point of collection (POC). There are no nationally accepted standards or cutoff concentrations for detecting drugs in oral fluids and for most analytes there are significant differences in cutoff concentrations across devices. Four devices were evaluated (OralLab), RapiScan, Drugwipe, and SalivaScreen) for their ability to meet manufacturers claims, and proposed federal standards for criminal justice and workplace programs. Human oral fluid fortified with known quantities of drug [drug(s) or metabolite(s)] was used to test these devices. Overall, the performance of these rapid POC oral fluid drug-testing devices was quite variable. Some devices performed well for the analysis of some drug classes but poorly for others. In general, most devices performed well for the detection of methamphetamine and opiates, but all performed poorly for the detection of cannabinoids. The ability to accurately and reliably detect cocaine and amphetamine was dependent on the individual device.

Equipment Design↗

PubMed2003

A liquid chromatographic-tandem mass spectrometric method for the analysis of serotonin and related indoles in human whole blood.

This paper describes a high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS-MS) method for the determination of serotonin (5-HT) and the related indoles tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid (IAA), and indole-3-proprionic acid (IPA) in whole blood. Ionization was achieved by atmospheric pressure chemical ionization (APCI). The method uses a solvent gradient to achieve baseline separation of all analytes. Linearity was established for all components from 10 to 500 ng/mL, with the exception of TRP, which was calibrated from 0.4 to 20 microg/mL. Limits of quantitation were established that were well below the normal endogenous concentrations of 5-HT and its related indoles. Recoveries of 5-HT, TRP, 5-HIAA, IAA, and IPA were 95%, 91%, 98%, 79%, and 65%, respectively, from whole blood matrix and varied less than 2%. Serotonin, TRP, and IAA results were verified by cross-validation with an external laboratory using a previously validated HPLC method with fluorometric detection. A regression analysis of the two data sets resulted in correlation coefficients of 0.981, 0.964, and 0.987 for 5-HT, TRP, and IAA, respectively.

Chromatography, High Pressure Liquid↗

PubMed2003

Capsaicinoids cause inflammation and epithelial cell death through activation of vanilloid receptors.

Capsaicinoids, found in less-than-lethal self-defense weapons, have been associated with respiratory failure and death in exposed animals and people. The studies described herein provide evidence for acute respiratory inflammation and damage to epithelial cells in experimental animals, and provide precise molecular mechanisms that mediate these effects using human bronchiolar and alveolar epithelial cells. Inhalation exposure of rats to pepper sprays (capsaicinoids) produced acute inflammation and damage to nasal, tracheal, bronchiolar, and alveolar cells in a dose-related manner. In vitro cytotoxicity assays demonstrated that cultured human lung cells (BEAS-2B and A549) were more susceptible to necrotic cell death than liver (HepG2) cells. Transcription of the human vanilloid receptor type-1, VR1 or TRPV1, was demonstrated by RT-PCR in all of these cells, and the relative transcript levels were correlated to cellular susceptibility. TRPV1 receptor activation was presumably responsible for cellular cytotoxicity, but prototypical functional antagonists of this receptor were cytotoxic themselves, and did not ameliorate capsaicinoid-induced damage. Conversely, the TRPV1 antagonist capsazepine, as well as calcium chelation by EGTA ablated cytokine (IL-6) production after capsaicin exposure. To address these seemingly contradictory results, recombinant human TRPV1 was cloned and overexpressed in BEAS-2B cells. These cells exhibited dramatically increased cellular susceptibility to capsaicinoids, measured using IL-6 production and cytotoxicity, and an apoptotic mechanism of cell death. Surprisingly, the cytotoxic effects of capsaicin in TRPV1 overexpressing cells were also not inhibited by TRPV1 antagonists or by treatments that modified extracellular calcium. Thus, capsaicin interacted with TRPV1 expressed by BEAS-2B and other airway epithelial cells to cause the calcium-dependent production of cytokines and, conversely, calcium-independent cell death. These results have demonstrated that capsaicinoids contained in pepper spray products produce airway inflammation and cause respiratory epithelial cell death. The mechanisms of these cellular responses to capsaicinoids appear to proceed via distinct cellular pathways, but both pathways are initiated by TRPV1.

Administration, Inhalation↗

PubMed2002

Determination of capsaicin, nonivamide, and dihydrocapsaicin in blood and tissue by liquid chromatography-tandem mass spectrometry.

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the analysis of capsaicin, nonivamide, and dihydrocapsaicin in blood and tissue has been developed. The method utilized a one-step liquid-liquid extraction that yielded an approximate 90% recovery of capsaicinoids from blood. Chomatographic separation of the capsaicinoids was achieved using a reversed-phase high-performance liquid chromatography column and a stepwise gradient of methanol and distilled water containing 0.1% (v/v) formic acid. Identification and quantitation of the capsaicinoids was achieved using electrospray ionization-tandem mass spectrometry monitoring the precursor-to-product-ion transitions for the internal standard octanoyl vanillamide (m/z 280 --> 137), capsaicin (m/z 306 --> 137), dihydrocapsaicin (m/z 308 -->137), and nonivamide (m/z 294 --> 137). Calibration curves, 1.0 to 250 ng/mL, were constructed by plotting concentration versus peak-area ratio (analyte/internal standard) and fitting the data with a weighted quadratic equation. The accuracy of the assay ranged from 90% to 107% for all analytes. The intra-assay precision (%RSD) for capsaicin was 4% at 2.5 ng/mL, 3% at 10 ng/mL, and 7% at 100 ng/mL. The interassay precision (% RSD) for capsaicin was 6% at 2.5 ng/mL, 6% at 10 ng/mL, and 7% at 100 ng/mL. Similar values for inter- and intra-assay precision were obtained for nonivamide and dihydrocapsaicin. This method was used to assay for capsaicinoids in blood and tissue samples collected from rats exposed to capsaicinoids via nose-only inhalation. The concentration of capsaicin in these samples ranged from < 1.0 to 90.4 ng/mL in the blood, < 5.0 to 167 pg/mg in the lung, and < 2.0 to 3.4 pg/mg in the liver.

Administration, Inhalation↗

PubMed2002

A field evaluation of five on-site drug-testing devices.

A field study was performed at two police agencies to evaluate the utility and accuracy of five on-site urine analysis drug-testing devices when used to test driving under the influence (DUI) arrestees. The devices evaluated were AccuSign, Rapid Drug Screen, TesTcup-5, TesTstik, and Triage. Standard workplace screening cut-off concentrations were used and samples were tested for marijuana, cocaine and metabolites, amphetamine(s), opiates, and PCP (except opiates 300 ng/mL). Four-hundred arrestees were recruited at each site, informed consent was obtained, and urine specimens were collected from each subject for analysis. Police officers conducted the testing with one device, and trained technicians performed testing with the other four devices. The device used by the officers was rotated. All positive and 5% of the negative samples were confirmed in a laboratory using mass spectrometry. Laboratory cut-off concentrations were 4 ng/mL for carboxy-THC; 50 ng/mL for benzoylecgonine; 100 ng/mL for amphetamines; 50 ng/mL for opiates; and 5 ng/mL for PCP. Approximately one-third (36%) of the subjects tested positive for at least one drug. No randomly selected sample, that tested negative on the devices, tested positive at the laboratory. Based on 800 specimens, the false-negative rate for each device was < 1% for all drug classes. A false positive was defined as testing positive with the device, but the specimen did not contain detectable drug, given the study reporting criteria. For marijuana, benzoylecgonine, and opiates, all devices had < or = 0.25% false-positive rates. For PCP, the false-positive rates were all < or = 1.5%. For amphetamine(s), the false-positive rates were all < or = 1.75%. These rates were adjusted because study confirmation batteries included methylenedioxyamphetamine, methylenedioxymethamphetamine (MDMA), additional over-the-counter sympathomimetic amines, hydromorphone, and hydrocodone. Without the expanded confirmation battery, false-positive rates approached 4% (Triage) for amphetamines and were > or = 2.25% for opiates. Fifty to 90% of the positive amphetamine(s) samples contained MDMA. A similar percentage of the opiate-positive samples contained hydromorphone or hydrocodone. When additional drugs were included in the confirmation testing, it was concluded that the on-site urine analysis drug-testing results were useful in DUI investigations.

Automobile Driving↗

PubMedDate not supplied

Assessment of pepper spray product potency in Asian and Caucasian forearm skin using transepidermal water loss, skin temperature and reflectance colorimetry.

Historically, pepper spray product potency has been established using a taste test evaluation. A taste test is subjective and may not be appropriate for assessing pepper potency in skin. The current study evaluated chemically diverse pepper sprays in human forearm skin using three objective, noninvasive parameters: transepidermal water loss, skin surface temperature and erythema, as a means for assessing dermal pharmacology, toxicology and product potency. Five commercial pepper spray products containing various capsaicinoid analogs at various concentrations were evaluated in duplicate on volar forearms of six Caucasians and six Asians using a 10 min exposure. Mean surface skin temperature, transepidermal water loss results were highly variable and therefore did not demonstrate dose responsive behavior to increasing capsaicinoid concentrations. Erythema, as measured by increases in a* (reflected light in the red-to-green color spectrum) of the L*a*b* uniform color scale, was superior among parameters evaluated in discriminating pepper spray potency and correlated well with the relative and total capsaicinoid concentration in the products. Products containing greater than 16 mg ml(-1) capsaicinoid concentration produced greater erythema responses in Caucasians than Asians. Asians responded greater to the synthetic analog, nonivamide, than to mixtures of capsaicinoids, while Caucasians responded equally to both capsaicinoid analogs. Thus, pepper spray product potency in human skin reflects the total capsaicinoid concentration, the specific capsaicin analog(s) present, and the race of the individual exposed. The finding that the reflectance colorimeter a* scale can differentiate these parameters in skin will have a significant impact on evaluating the use and efficacy of pepper spray products in humans.

Adult↗

PubMedDate not supplied

Analysis of the nutritional supplement 1AD, its metabolites, and related endogenous hormones in biological matrices using liquid chromatography-tandem mass spectrometry.

1,5alpha-Androsten-3beta,17beta-diol and/or 1,5alpha-androsten-3,17-dione (1AD) is an over-the-counter pro-hormone nutritional supplement designed to enhance strength and performance in athletes. 1AD purportedly mimics the pharmacological activity of testosterone through activation of the pro-hormones to their active form 1,5alpha-androsten-17beta-ol-3-one or Delta(1)-testosterone. This testosterone analogue ostensibly possesses strong androgenic potency without the adverse effects associated with aromatization of testosterone to estrogens. We have developed a highly sensitive and selective liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of 1AD, its analogues, and several structurally related endogenous hormones in plasma and urine. The limits of quantitation for the analytes ranged from 0.25 to 0.5 ng/mL. The accuracy of the assay was 92-113% with a precision of 2-12% relative standard deviation (RSD) for all analytes at 1.0, 5.0, and 15.0 ng/mL, respectively. The interassay precision was 6-16% RSD, and the accuracy was 90-105%. We have used this assay to determine the unconjugated and total (conjugated and unconjugated) concentrations of 1AD, its analogues, androstenediol, androstenedione, testosterone, dihydrotestosterone, and estradiol, in plasma and urine, as well as to investigate the metabolic fate of the three 1AD analogues (diol, dione, and active forms) when incubated with rat liver microsomes or rat testicular homogenates. Concentrations of both unconjugated and total testosterone in plasma were approximately 1.5 ng/mL and ranged from undetectable to 4.1 ng/mL in urine. 1AD and its analogues were not detected in plasma or urine. In vitro metabolism experiments using rat liver microsomes and testicular homogenates provided evidence for the interconversion of the three 1AD analogues, biosynthesis, and decomposition of several endogenous hormones, as well as evidence for 1AD analogue-induced changes in the typical profiles of testosterone and androstenedione in testicular tissue.

Anabolic Agents↗

PubMedDate not supplied

An evaluation of selected oral fluid point-of-collection drug-testing devices.

Point-of-collection oral fluids drug-testing devices are being marketed for a variety of medico-legal purposes where they may complement existing technologies and be used to detect drugs following recent ingestion. To assess the utility of these devices for use in drugged-driving investigations, we performed a laboratory evaluation of four devices and those results were published previously. In the study reported here, two more devices, Oratect(R) (Branan) and Uplink(R) (OraSure), were evaluated for their ability to detect amphetamines, cocaine, opiates, and cannabinoids. An additional device, Drugwipe (Securtec), was evaluated for the detection of cocaine and cannabinoids. Each of the devices was assessed for their ability to meet the manufacturers' claimed cutoff concentrations and to meet cutoffs proposed for federal workplace programs. In general, the Branan and OraSure devices detected amphetamine, methamphetamine, opiates, and cannabinoid metabolite (THC-COOH) well in the concentration ranges approximating those proposed by the Substance Abuse and Mental Health Services Administration (SAMHSA), but all three devices performed poorly in detecting Delta9-tetrahydrocannabinol (THC) at the proposed SAMHSA cutoff. The ability to accurately and reliably detect cocaine was dependent on the individual device, and the Branan and Securetec devices were more effective than OraSure at detecting parent cocaine.

Equipment Design↗