Driving Under the Influence of Cannabis: Impact of Combining Toxicology Testing with Field Sobriety Tests.

BACKGROUND: Cannabis is increasingly used both medically and recreationally. With widespread use, there is growing concern about how to identify cannabis-impaired drivers. METHODS: A placebo-controlled randomized double-blinded protocol was conducted to study the effects of cannabis on driving performance. One hundred ninety-one participants were randomized to smoke ad libitum a cannabis cigarette containing placebo or delta-9-tetrahydrocannabinol (THC) (5.9% or 13.4%). Blood, oral fluid (OF), and breath samples were collected along with longitudinal driving performance on a simulator (standard deviation of lateral position [SDLP] and car following [coherence]) over a 5-hour period. Law enforcement officers performed field sobriety tests (FSTs) to determine if participants were impaired. RESULTS: There was no relationship between THC concentrations measured in blood, OF, or breath and SDLP or coherence at any of the timepoints studied (P > 0.05). FSTs were significant (P < 0.05) for classifying participants into the THC group vs the placebo group up to 188 minutes after smoking. Seventy-one minutes after smoking, FSTs classified 81% of the participants who received active drug as being impaired. However, 49% of participants who smoked placebo (controls) were also deemed impaired at this same timepoint. Combining a 2 ng/mL THC cutoff in OF with positive findings on FSTs reduced the number of controls classified as impaired to zero, 86 minutes after smoking the placebo. CONCLUSIONS: Requiring a positive toxicology result in addition to the FST observations substantially improved the classification accuracy regarding possible driving under the influence of THC by decreasing the percentage of controls classified as impaired.

Driving Performance and Cannabis Users' Perception of Safety: A Randomized Clinical Trial.

IMPORTANCE: Expanding cannabis medicalization and legalization increases the urgency to understand the factors associated with acute driving impairment. OBJECTIVE: To determine, in a large sample of regular cannabis users, the magnitude and time course of driving impairment produced by smoked cannabis of different Δ9-tetrahydrocannabinol (THC) content, the effects of use history, and concordance between perceived impairment and observed performance. DESIGN, SETTING, AND PARTICIPANTS: This double-blind, placebo-controlled parallel randomized clinical trial took place from February 2017 to June 2019 at the Center for Medicinal Cannabis Research, University of California San Diego. Cannabis users were recruited for this study, and analysis took place between April 2020 and September 2021. INTERVENTIONS: Placebo or 5.9% or 13.4% THC cannabis smoked ad libitum. MAIN OUTCOMES AND MEASURES: The primary end point was the Composite Drive Score (CDS), which comprised key driving simulator variables, assessed prior to smoking and at multiple time points postsmoking. Additional measures included self-perceptions of driving impairment and cannabis use history. RESULTS: Of 191 cannabis users, 118 (61.8%) were male, the mean (SD) age was 29.9 (8.3) years, and the mean (SD) days of use in the past month was 16.7 (9.8). Participants were randomized to the placebo group (63 [33.0%]), 5.9% THC (66 [34.6%]), and 13.4% THC (62 [32.5%]). Compared with placebo, the THC group significantly declined on the Composite Drive Score at 30 minutes (Cohen d = 0.59 [95% CI, 0.28-0.90]; P < .001) and 1 hour 30 minutes (Cohen d = 0.55 [95% CI, 0.24-0.86]; P < .001), with borderline differences at 3 hours 30 minutes (Cohen d = 0.29 [95% CI, -0.02 to 0.60]; P = .07) and no differences at 4 hours 30 minutes (Cohen d = -0.03 [95% CI, -0.33 to 0.28]; P = .87). The Composite Drive Score did not differ based on THC content (likelihood ratio χ24 = 3.83; P = .43) or use intensity (quantity × frequency) in the past 6 months (likelihood ratio χ24 = 1.41; P = .49), despite postsmoking blood THC concentrations being higher in those with the highest use intensity. Although there was hesitancy to drive immediately postsmoking, increasing numbers (81 [68.6%]) of participants reported readiness to drive at 1 hour 30 minutes despite performance not improving from initial postsmoking levels. CONCLUSIONS AND RELEVANCE: Smoking cannabis ad libitum by regular users resulted in simulated driving decrements. However, when experienced users control their own intake, driving impairment cannot be inferred based on THC content of the cigarette, behavioral tolerance, or THC blood concentrations. Participants' increasing willingness to drive at 1 hour 30 minutes may indicate a false sense of driving safety. Worse driving performance is evident for several hours postsmoking in many users but appears to resolve by 4 hours 30 minutes in most individuals. Further research is needed on the impact of individual biologic differences, cannabis use history, and administration methods on driving performance. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02849587.

Blood and Oral Fluid Cannabinoid Profiles of Frequent and Occasional Cannabis Smokers.

Increased prevalence of cannabis consumption and impaired driving are a growing public safety concern. Some states adopted per se driving laws, making it illegal to drive with more than a specified blood concentration of ∆9-tetrahydrocannabinol (THC) in a biological fluid (typically blood). Blood THC concentrations decrease significantly (∼90%) with delays in specimen collection, suggesting the use of alternative matrices, such as oral fluid (OF). We characterized 10 cannabinoids' concentrations, including THC metabolites, in blood and OF from 191 frequent and occasional users by liquid chromatography with tandem mass spectrometry for up to 6 h after ad libitum smoking. Subjects self-titrated when smoking placebo, 5.9 or 13.4% THC cannabis. Higher maximum blood THC concentrations (Cmax) were observed in individuals who received the 5.9% THC versus the 13.4% THC plant material. In blood, the Cmax of multiple analytes, including THC and its metabolites, were increased in frequent compared to occasional users, whereas there were no significant differences in OF Cmax. Blood THC remained detectable (≥5 ng/mL) at the final sample collection for 14% of individuals who smoked either the 5.9 or 13.4% THC cigarette, whereas 54% had detectable THC in OF when applying the same cutoff. Occasional and frequent cannabis users' profiles were compared, THC was detectable for significantly longer duration in blood and OF from frequent users. Detection rates between frequent and occasional users at multiple per se cutoffs showed larger differences in blood versus OF. Understanding cannabinoid profiles of frequent and occasional users and the subsequent impact on detectability with current drug per se driving limits is important to support forensic interpretations and the development of scientifically supported driving under the influence of cannabis laws.

Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect cannabinoids in whole blood and breath.

Background The widespread availability of cannabis raises concerns regarding its effect on driving performance and operation of complex equipment. Currently, there are no established safe driving limits regarding ∆9-tetrahydrocannabinol (THC) concentrations in blood or breath. Daily cannabis users build up a large body burden of THC with residual excretion for days or weeks after the start of abstinence. Therefore, it is critical to have a sensitive and specific analytical assay that quantifies THC, the main psychoactive component of cannabis, and multiple metabolites to improve interpretation of cannabinoids in blood; some analytes may indicate recent use. Methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to quantify THC, cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), (±)-11-nor-9-carboxy-Δ9-THC (THCCOOH), (+)-11-nor-Δ9-THC-9-carboxylic acid glucuronide (THCCOOH-gluc), cannabigerol (CBG), and tetrahydrocannabivarin (THCV) in whole blood (WB). WB samples were prepared by solid-phase extraction (SPE) and quantified by LC-MS/MS. A rapid and simple method involving methanol elution of THC in breath collected in SensAbues® devices was optimized. Results Lower limits of quantification ranged from 0.5 to 2 µg/L in WB. An LLOQ of 80 pg/pad was achieved for THC concentrations in breath. Calibration curves were linear (R2>0.995) with calibrator concentrations within ±15% of their target and quality control (QC) bias and imprecision ≤15%. No major matrix effects or drug interferences were observed. Conclusions The methods were robust and adequately quantified cannabinoids in biological blood and breath samples. These methods will be used to identify cannabinoid concentrations in an upcoming study of the effects of cannabis on driving.

Proteomic screening of plasma identifies potential noninvasive biomarkers associated with significant/advanced fibrosis in patients with nonalcoholic fatty liver disease.

Noninvasive biomarkers are clinically useful for evaluating liver fibrosis stage in patients with nonalcoholic fatty liver disease (NAFLD). The aim of the present study was to compare plasma proteins in patients with early nonalcoholic steatohepatitis (NASH) (F0-F1) versus NASH with significant/advanced fibrosis (F2-F4) to determine whether candidate proteins could be used as potential noninvasive biomarkers. Nineteen biopsy-proven NAFLD patients including ten early NASH patients and nine NASH patients with significant/advanced fibrosis were enrolled in the present study. High-resolution proteomics screening of plasma was performed with the SCIEX TripleTOF 5600 System. Proteins were quantified using two different software platforms, Progenesis Qi and Scaffold Q+, respectively. Progenesis Qi analysis resulted in the discovery of 277 proteins compared with 235 proteins in Scaffold Q+. Five consensus proteins (i.e. Complement component C7; α-2-macroglobulin; Complement component C8 γ chain; Fibulin-1; α-1-antichymotrypsin) were identified. Complement component C7 was three-fold higher in the NASH group with significant/advanced fibrosis (F2-F4) compared with the early NASH (F0-F1) group (q-value = 3.6E-6). Complement component C7 and Fibulin-1 are positively correlated with liver stiffness (P=0.000, P=0.002, respectively); whereas, Complement component C8 γ chain is negatively correlated (P=0.009). High levels of Complement C7 are associated with NASH with significant/advanced fibrosis and Complement C7 is a perfect classifier of patients included in this pilot study. Further studies will be needed in a larger validation cohort to confirm the utility of complement proteins as biomarkers or mechanistic determinants of NASH with significant/advanced fibrosis.

Validation of a liquid chromatography-tandem mass spectrometry method for analyzing cannabinoids in oral fluid.

A liquid chromatography tandem mass spectrometry method was developed for quantifying ten cannabinoids in oral fluid (OF). This method utilizes OF collected by the Quantisal™ device and concurrently quantifies cannabinol (CBN), cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-THC (11-OH-THC), 11-nor-9-carboxy-Δ9-THC (THC-COOH), 11-nor-9-carboxy-Δ9-THC glucuronide (THC-COOH-gluc), Δ9-THC glucuronide (THC-gluc), cannabigerol (CBG), tetrahydrocannabiverin (THCV), and Δ9-tetrahydrocannabinolic acid A (THCA-A). Solid phase extraction was optimized using Oasis Prime HLB 30 mg 96-well plates. Cannabinoids were separated by liquid chromatography over a BEH C18 column and detected by a Waters TQ-S micro tandem mass spectrometer. The lower limits of quantification (LLOQ) were 0.4 ng/mL for CBN, CBD, THC, 11-OH-THC, THC-gluc, and THCV; and 1.0 ng/mL for THC-COOH, THC-COOH-gluc, CBG and THCA-A. Linear ranges extended to 2000 ng/mL for THC and 200 ng/mL for all other analytes. Inter-day analytical bias and imprecision at three levels of quality control (QC) was within ±15%. Mean extraction efficiencies ranged from 26.0-98.8%. Applicability of this method was tested using samples collected from individuals randomly assigned to smoke either a joint containing <0.1%, 5.9%, or 13.4% THC content. This method was able to identify and calculate the concentration of 6 of 10 cannabinoids validated in this method.

Multicenter assessment of a hemoglobin A1c point-of-care device for diagnosis of diabetes mellitus.

OBJECTIVE: A multisite investigation compared the analytical performance of a point-of-care (POC) HbA1c device with multiple commonly used HbA1c laboratory methods and an NGSP (National Glycohemoglobin Standardization Program) reference method. RESEARCH DESIGN AND METHODS: The Afinion AS100 POC device analyzed HbA1c using 618 EDTA whole blood excess patient specimens with clinically indicated HbA1c testing. Results were compared to measurements across five clinical laboratories and the NGSP reference method. Precision was evaluated over 8-10 consecutive days for low-, mid-, and high-range HbA1c specimens at all five sites. RESULTS: Over a wide range of HbA1c values (4.0%-15% HbA1c), 97.1% of the POC results and 94.5% of routine laboratory results fell within the target value of ±6% of the NGSP reference method results. The POC HbA1c results at 6.5% exhibited a total relative bias of -0.6% (-0.04% HbA1c) compared to the reference method while the aggregate of laboratory methods displayed a relative bias of -0.9% (-0.06% HbA1c). The total imprecision of the POC results ranged from 0.74-2.13% CV across the analytic measurement range compared to 0.81-3.23% CV for the routine laboratory methods. CONCLUSIONS: The accuracy and precision of the Afinion POC HbA1c method was comparable to the laboratory HbA1c methods supporting the FDA's recent approval of the Afinion HbA1c Dx device for use in the diagnosis of diabetes.

Interpretation of Pain Management Testing Results Using Case Examples.

BACKGROUND: Because of the increasing volume of opiate-related overdoses, clinical testing of urine for drugs and related compounds in pain management clinics has become increasingly important. Interpreting findings of drugs present in urine specimens requires knowledge of pharmacokinetics, metabolism, drug purity, and cutoff concentrations used to report a positive result. CONTENT: This case-based mini-review provides examples of how to interpret immunoassay and quantitative confirmatory urine drug-testing results. Particular emphasis is placed on interpretation of opiate and benzodiazepine results, as these drugs have complicated metabolic profiles. SUMMARY: Both determining patient medication compliance and identifying the presence of additional drugs provides important information to the treating physician involved in managing pain. Mass spectrometry-based methods are required to identify specific drugs present and can provide important quantitative data for interpreting opiate medication compliance.

Feeding a Modified Fish Diet to Bottlenose Dolphins Leads to an Increase in Serum Adiponectin and Sphingolipids.

Feeding a modified fish diet has been suggested to improve insulin sensitivity in bottlenose dolphins; however, insulin sensitivity was not directly measured. Since demonstrating an improvement in insulin sensitivity is technically difficult in dolphins, we postulated that directional changes in the hormone axis: fibroblast growth factor 21 (FGF21)/Adiponectin/Ceramide (Cer), could provide further support to this hypothesis. We measured 2-h post-prandial serum FGF21, total adiponectin, percent unmodified adiponectin, ceramide, and sphingosine levels from dolphins fed a diet rich in heptadecanoic acid (C17:0) over 24 weeks. Serum FGF21 was quantified by ELISA with an observed range of 129-1599 pg/ml, but did not significantly change over the 24-week study period. Total adiponectin levels (mean ± SD) significantly increased from 776 ± 400 pmol/ml at week 0 to 1196 ± 467 pmol/ml at week 24. The percent unmodified adiponectin levels (mean ± SD) decreased from 23.8 ± 6.0% at week 0 to 15.2 ± 5.2% at week 24. Interestingly, although FGF21 levels did not change, there was a good correlation between FGF21 and total adiponectin (ρ = 0.788, P < 0.001). We quantified the abundances of serum ceramides and sphingosines (SPH) because adiponectin has a defined role in sphingolipid metabolism through adiponectin receptor-mediated activation of ceramidases. The most abundant ceramide in dolphin sera was Cer 24:1 comprising 49% of the ceramides measured. Significant reductions were observed in the unsaturated Cer 18:1, Cer 20:1, and Cer 24:1, whereas significant increases were observed in saturated Cer 22:0, Cer 24:0, and Cer 26:0. However, total serum ceramides did not change. Significant elevations were detected for total sphingosine, dihydrosphingosine, sphingosine-1-phosphate, and dihydrosphingosine-1-phosphate. Proteomic analysis of the serum proteins revealed few changes in serum proteins over the study period. In conclusion, shifting the dolphin diet to fishes rich in odd chain saturated fatty acids, such as C17:0, resulted in increased serum levels of the insulin sensitizing hormone adiponectin and serum SPH consistent with an insulin-sensitizing phenotype. It is still unclear whether FGF21 plays a role in the regulation of adiponectin in dolphins, similar to that shown in laboratory animal models.

Regulation of the tumor suppressor FOXO3 by the thromboxane-A2 receptors in urothelial cancer.

The transcription factor FOXO3 is a well-established tumor suppressor whose activity, stability, and localization are regulated by phosphorylation and acetylation. Previous data by our laboratory demonstrated amplified thromboxane-A2 signaling was associated with poor prognoses in bladder cancer patients and overexpression of the thromboxane-A2 isoform-ß receptor (TPß), but not TPα, induced malignant transformation of immortalized bladder cells in vivo. Here, we describe a mechanism of TP mediated modulation of FOXO3 activity and localization by phosphorylation and deacetylation in a bladder cancer cell model. In vitro gain and loss of function studies performed in non-transformed cell lines, UROsta and SV-HUC, revealed knockdown of FOXO3 expression by shRNA increased cell migration and invasion, while exogenously overexpressing TPß raised basal phosphorylated (p)FOXO3-S294 levels. Conversely, overexpression of ERK-resistant, mutant FOXO3 reduced increases in UMUC3 cell migration and invasion, including that mediated by TP agonist (U46619). Additionally, stimulation of UMUC3 cells with U46619 increased pFOXO3-S294 expression, which could be attenuated by treatment with a TP antagonist (PTXA2) or ERK inhibitor (U0126). Initially U46619 caused nuclear accumulation of pFOXO3-S294; however, prolonged stimulation increased FOXO3 cytoplasmic localization. U46619 stimulation decreased overall FOXO3 transcriptional activity, but was associated with increased expression of its pro-survival target, manganese superoxide dismutase. The data also shows that TP stimulation increased the expression of the histone deacetylase, SIRT1, and corresponded with decreased acetylated-FOXO3. Collectively, the data suggest a role for TP signaling in the regulation of FOXO3 activity, mediated in part through phosphorylation and deacetylation.