Roy Gerona

CAPS Acts at a Prefusion Step in Dense-Core Vesicle Exocytosis as a PIP2 Binding Protein

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.

Synthetic Cannabinoid–Related Illnesses and Deaths

Use of synthetic cannabinoids (SCs) in the United States is increasing, as are clusters of cases of serious adverse health effects, including death. Though SC intoxication can be difficult to identify, some steps can be taken to respond more quickly to future outbreaks.

Role of liquid chromatography–high-resolution mass spectrometry (LC-HR/MS) in clinical toxicology

Background. Gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) are widely used to confirm drug screening results and for urine screening in presumed intoxicated patients. These techniques are better suited to targeted analysis than to general unknown screening and, due to the complexity of testing, results are seldom available rapidly enough to contribute to the immediate care of the patient. High resolution (HR)/MS with time-of-flight (TOF) or orbitrap instruments offer potential advantages in clinical toxicology. Comparison of GC-MS, LC-MS/MS and LC-HR/MS. For unknown analyses, GC-MS and LC-MS/MS require comparison of full-scan spectra against preestablished libraries. Operation in full-scan mode greatly reduces sensitivity and some drugs present in low but significant concentrations may be missed. Selected ion monitoring (SIM) in GC/MS and selected reaction monitoring (SRM) in LC-MS/MS, where only targeted ions are monitored, increase sensitivity but require prior knowledge of what compound is to be measured. LC-HR/MS offers mass assignment with an accuracy of 0.001 atomic mass units (amu) compared with 1 amu in conventional MS. Tentative identification is thus directed to a very limited set of compounds (or even one unique compound) based on the exact molecular formula rather than a fragmentation pattern, since HR/MS can discriminate between compounds with the same nominal molecular mass. LC-MS/MS has clear advantages over GC/MS in ease and speed of sample preparation and the opportunities for its automation. LC-HR/MS is more suitable to clinical toxicology because the drugs present in a sample are rarely known a priori, and tentative identifications of unknowns can be made without the availability of a reference standard or a library spectrum. Blood can be used in preference to urine which is more relevant to the patients current clinical situation. Methods. A literature search was conducted using PUBMED for clinical toxicology, adulterants in illicit drugs and herbal supplements, and case reports using LC-TOF/MS and LC-HR/MS. Only 42 papers in English were identified in these searches. LC-HR/MS in clinical toxicology. LC-HR/MS has been used to detect designer drugs, doping agents, (neurosteroids) and adulterants such as levamisole, a veterinary antihelmitic found in street cocaine, and pharmaceuticals in herbal medications marketed to contain only natural ingredients. LC-HR/MS has proved useful for cases where existing tests were unable to identify the cause of the intoxication. One patient suffered a drug-induced seizure which was originally thought to be caused by an herbal medication, but diphenhydramine was determined to be the culprit. In another, 5-oxoproline was identified as the cause of metabolic acidosis seen in chronic acetaminophen (paracetamol) use. LC-HR/MS has successfully identified medications that were mislabeled or misrepresented street drugs. In one case, medications sold as diazepam were determined to be glyburide instead. The identification of novel designer amines, stimulants found in “bath salts”, and synthetic cannabinoids are well suited to LC-HR/MS. Dozens or even hundreds of possible compounds cannot realistically be tested on an individual basis by targeted LC-MS/MS or GC/MS analysis. Conclusions. LC-HR/MS offers unique opportunities for time-sensitive clinical analysis of blood samples from intoxicated patients and for comprehensive screening in a wide range of situations and materials. While the identification is not as definitive as that obtained by conventional fragmentation MS, the presumptive identification can be confirmed later with standards and spectral library matches. Optimum utilization of the presumptive diagnosis requires close collaboration between the laboratory analysts and their clinical counterparts.

Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca(2+) is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca(2+) also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca(2+)-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca(2+)-dependent SNARE binding or in Ca(2+)-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca(2+)-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca(2+)-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca(2+)-dependent membrane insertion and SNARE binding to drive fusion pore expansion.

Bisphenol-A (BPA), BPA glucuronide, and BPA sulfate in midgestation umbilical cord serum in a northern and central California population.

Bisphenol-A (BPA) is an endocrine disrupting chemical used in numerous consumer products, resulting in universal exposure in the United States. Prenatal exposure to BPA is associated with numerous reproductive and developmental effects in animals. However, little is known about human fetal exposure or metabolism of BPA during midgestation. In the present study, we present a new liquid chromatography-tandem mass spectrometry method to directly measure concentrations of BPA and two predominant metabolic conjugates-BPA glucuronide and BPA sulfate-in umbilical cord serum collected from elective second trimester pregnancy terminations. We detected at least one form of BPA in all umbilical cord serum samples: BPA (GM 0.16, range <LOD-52.26 ng/mL), BPA glucuronide (GM 0.14, range <LOD-5.41 ng/mL) and BPA sulfate (GM 0.32, range <LOD-12.65 ng/mL). Levels of BPA ranged from less than 1/100th to over 400 times higher than levels of BPA in conjugated form. Although levels of BPA in conjugated form exceeded BPA levels in about 3/4 of the samples, BPA levels were higher in samples with total BPA above the median. Our findings suggest universal fetal exposure to BPA in our study population, with some at relatively high levels, and we provide the first evidence of detectable BPA sulfate in midgestation fetuses.

Acute kidney injury associated with smoking synthetic cannabinoid

Abstract Context and objectives. Synthetic cannabinoids are illegal drugs of abuse known to cause adverse neurologic and sympathomimetic effects. They are an emerging health risk: 11% of high school seniors reported smoking them during the previous 12 months. We describe the epidemiology of a toxicologic syndrome of acute kidney injury associated with synthetic cannabinoids, review the toxicologic and public health investigation of the cluster, and describe clinical implications of the cluster investigation. Materials and methods. Case series of nine patients affected by the toxicologic syndrome in Oregon and southwestern Washington during May–October 2012. Cases were defined as acute kidney injury (creatinine > 1.3 mg/dL) among persons aged 13–40 years without known renal disease who reported smoking synthetic cannabinoids. Toxicology laboratories used liquid chromatography and time-of-flight mass spectrometry to test clinical and product specimens for synthetic cannabinoids, their metabolites, and known nephrotoxins. Public health alerts informed clinicians, law enforcement, and the community about the cluster and the need to be alert for toxidromes associated with emerging drugs of abuse. Results. Patients were males aged 15–27 years (median, 18 years), with intense nausea and flank or abdominal pain, and included two sets of siblings. Peak creatinine levels were 2.6–17.7 mg/dL (median, 6.6 mg/dL). All patients were hospitalized; one required dialysis; none died. No alternate causes of acute kidney injury or nephrotoxins were identified. Patients reported easily purchasing synthetic cannabinoids at convenience, tobacco, and adult bookstores. One clinical and 2 product samples contained evidence of a novel synthetic cannabinoid, XLR-11 ([1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone). Discussion and conclusion. Whether caused by direct toxicity, genetic predisposition, or an as-yet unidentified nephrotoxin, this association between synthetic cannabinoid exposure and acute kidney injury reinforces the need for vigilance to detect new toxicologic syndromes associated with emerging drugs of abuse. Liquid chromatography and time-of-flight mass spectrometry are useful tools in determining the active ingredients in these evolving products and evaluating them for toxic contaminants.

Synthetic cannabinoid use associated with acute kidney injury

The use of synthetic cannabinoids has been associated with multiple adverse effects, including seizures and psychosis. 1 Possible nephrotoxic effects from synthetic cannabinoid use have recently been reported. 2 We report a case of acute kidney injury (AKI) associated with the use of a synthetic cannabinoid product. A previously healthy 26-year-old male was presented to the emergency department with one day of abdominal pain, nausea, vomiting and lower back pain. His vital signs on arrival demonstrated a temperature of 97.7 ° F, heart rate of 54 beats per minute, blood pressure 151/40 mmHg, and respiratory rate of 16 breaths per minute with a SatO 2 of 100% on room air. His physical exam was unremarkable. He denied the use of ibuprofen or any other medication. He also denied using any herbal medications. He admitted to regular tobacco use but denied alcohol use. He did admit to smoking a product that he had obtained via the internet called “ Mr. Happy ” . He stated that he used this product two or three times a day for approximately one year and had used the product on the morning of his presentation. He denied any prior episodes of pain, vomiting, or agitation with the use of this product. Laboratory evaluation proved to be remarkable for a 14.4 K/mm3 WBC, 5.38 mg/dL serum creatinine, 30 mg/dL blood urea nitrogen (BUN), and urinalysis with 1+ protein and trace blood. His hemoglobin, platelets, electrolytes and total creatine kinase (CK) were normal. A urine immunoassay drug screen turned out to be negative. He was admitted to the hospital for further evaluation. The results of an antinuclear antibody titer turned out to be negative. A renal ultrasound was interpreted as normal and a renal biopsy demonstrated rare globally sclerotic glomeruli without evidence of acute glomerular disease. On Day 2 in the hospital, his creatinine and BUN peaked at 7.74 and 39 mg/dL, respectively. He was discharged after six days in the hospital with AKI of unknown etiology and serum creatinine of 3.09 mg/dL. Twenty three days later his serum creatinine was 1.1 mg/dL. He denied any further use of “ Mr. Happy ” or similar products. An initial urine sample, two serial serum samples, and the “ Mr. Happy ” product were tested with liquid chromatographytime-of-fl ight mass spectrometry (LC-TOF/MS) (TOF 6230, LC 1260, Agilent) for 214 pharmaceuticals and drugs of abuse, 40 synthetic cannabinoids and 16 synthetic cathinones, and 3 aminoindanes. Only XLR-11, UR-144, and their common metabolite, XLR-11 Npentanoic acid were identifi ed. Table 1 details the results of the LC-TOF/MS screen. XLR-11 and UR-144 are synthetic cannabinoids that have recently been detected in “ legal high ” products. 3 XLR-11 is the fl uorinated analog of UR-144 and they share a common metabolite, XLR-11 N-pentanoic acid. They are reported to be cannabinoid-2 receptor agonists. 4 There are no reports describing their effects in humans. While multiple adverse effects have been associated with synthetic cannabinoid use, AKI has only been noted in one prior case series. 2 However, that series is limited by the fact that no synthetic cannabinoids were identifi ed in biological or product specimens. While heavy marijuana use has been cited to cause glomerulonephritis, it is unclear how or if XLR-11 or UR-144 are contributing to AKI. 5 The presence of fl uorine in XLR-11 may predispose it or its metabolites to nephrotoxic effects, perhaps similar to fl uorinated anesthetics. 6 It is also possible that this product contained a nephrotoxic plant material or that an unidentifi ed chemical created from the burning of these products may have been responsible. This case report is limited by the fact that these “ legal high ” products are known to vary greatly in their composition over time. 7 Therefore the compounds we identifi ed in this particular “ Mr. Happy ” product and in the patient may differ from those that this patient was exposed to over the past year. Though we have no prior product sample to test, it is likely that the composition or concentration of the synthetic cannabinoids in the “ Mr. Happy ” product evolved over time. This could explain why this patient did not develop AKI earlier despite prolonged use of this product. Furthermore, while we can confi rm recent exposure, we cannot quantify this patient ’ s exposure to this product over the last year. Finally, it remains possible that this patient ’ s AKI and his exposure to XLR-11 and UR-144 are coincidental. This case suggests an association between the use of these synthetic cannabinoids and AKI but further studies examining possible mechanism of injury are needed. Until further information is available, it may be prudent for health care providers to consider AKI as a possible complication from the use of XLR-11 and UR-144.

“Zombie” Outbreak Caused by the Synthetic Cannabinoid AMB-FUBINACA in New York

BACKGROUND New psychoactive substances constitute a growing and dynamic class of abused drugs in the United States. On July 12, 2016, a synthetic cannabinoid caused mass intoxication of 33 persons in one New York City neighborhood, in an event described in the popular press as a “zombie” outbreak because of the appearance of the intoxicated persons. METHODS We obtained and tested serum, whole blood, and urine samples from 8 patients among the 18 who were transported to local hospitals; we also tested a sample of the herbal “incense” product “AK‐47 24 Karat Gold,” which was implicated in the outbreak. Samples were analyzed by means of liquid chromatography–quadrupole time‐of‐flight mass spectrometry. RESULTS The synthetic cannabinoid methyl 2‐(1‐(4‐fluorobenzyl)‐1H‐indazole‐3‐carboxamido)‐3‐methylbutanoate (AMB‐FUBINACA, also known as MMB‐FUBINACA or FUB‐AMB) was identified in AK‐47 24 Karat Gold at a mean (±SD) concentration of 16.0±3.9 mg per gram. The de‐esterified acid metabolite was found in the serum or whole blood of all eight patients, with concentrations ranging from 77 to 636 ng per milliliter. CONCLUSIONS The potency of the synthetic cannabinoid identified in these analyses is consistent with strong depressant effects that account for the “zombielike” behavior reported in this mass intoxication. AMB‐FUBINACA is an example of the emerging class of “ultrapotent” synthetic cannabinoids and poses a public health concern. Collaboration among clinical laboratory staff, health professionals, and law enforcement agencies facilitated the timely identification of the compound and allowed health authorities to take appropriate action.

‘Crazy Monkey’ Poisons Man and Dog: Human and canine seizures due to PB-22, a novel synthetic cannabinoid

Abstract Context. Synthetic cannabinoids have been manufactured, sold, and consumed for recreational purposes since at least 2004; their use has been associated with adverse psychiatric, cardiovascular, renal, pulmonary, and neurologic effects. We report simultaneous canine and human clinical cases associated with exposure to a novel synthetic cannabinoid, PB-22 (QUPIC). Case report. A 22-year-old man brought his dog to a veterinary clinic after it had a seizure. During the course of the canines evaluation, the human patient was witnessed to have a generalized tonic–clonic seizure. He was intubated for agitation and combativeness after his arrival to the emergency department (ED). He was extubated the next day without discernable sequelae. The canine patient received intravenous hydration and was also discharged to home after a period of close observation. The man later endorsed smoking three containers of a substance called “Crazy Monkey” daily for several weeks, but would not disclose how his dog had been exposed. The convulsant effects of “Crazy Monkey” were confirmed in this patient when, three months later, he was sedated, paralyzed, intubated, and admitted to another local hospital for seizures in the context of smoking the same product. Discussion. Laboratory analysis of samples obtained from the human and canine patients. A sample of the product (labeled “Crazy Monkey”) revealed the presence of PB-22 (QUPIC), a novel synthetic cannabinoid. Additionally, serum and urine samples from the human patient contained metabolites of a second compound, UR-144. Conclusion. We present a laboratory-confirmed case report of human and canine neurotoxicity associated with a novel synthetic cannabinoid, PB-22 (QUIPIC).

Multiple MDMA (Ecstasy) Overdoses at a Rave Event: A Case Series

Twelve patients with 3,4-methylenedioxymethamphetamine (MDMA) toxicity from a single rave event presented to multiple San Francisco Bay area hospitals with various life-threatening complications including seizures and hyperthermia. Eight required emergent endotracheal intubation and six had hypotension. Hyperkalemia, acute kidney injury, and rhabdomyolysis were present in most of the patients. In all, 2 patients died, 4 survived with permanent neurologic, musculoskeletal, and/or renal sequelae, and 6 survived without any apparent lasting deficits. Hyperthermia was present in 10 patients and was severe (40.9-43° C) in 7. Using multiple cooling methods, the average time to achieve cooling was 2.7 hours. Serum drug analysis was performed on 3 patients, demonstrating toxic MDMA concentrations without the presence of other xenobiotics. Two capsules confiscated by police at the event contained 82% and 98% MDMA, respectively, without other pharmacologically active compounds. Capsule #2 contained 270 mg MDMA, which is more than twice the amount of MDMA usually contained in 1 dose. The MDMA-induced hyperthermia significantly contributed to the morbidity and mortality in this case series. Factors contributing to the severity of the hyperthermia include ingestion of large doses of MDMA, a warm ambient environment, and physical exertion.