Quantitation of propofol metabolites by LC-MS/MS demonstrating long detection window for urine drug monitoring.

Introduction: Chromatographic methods for analysis of propofol and its metabolites have been widely used in pharmacokinetic studies of propofol distribution, metabolism, and clearance. Application of chromatographic methods is also needed in clinical and forensic laboratories for detecting and monitoring propofol misuse. Objective: We report a method for sensitive analysis of propofol, propofol 1-glucuronide (PG), 4-hydroxypropofol 1-glucuronide (1-QG), 4-hydroxypropofol 4-glucuronide (4-QG) and 4-hydroxypropofol 4-sulfate (4-QS) in urine by LC-MS/MS analysis. The method employs a simple dilute-and-analyze sample preparation with stable isotope internal standardization. Results: Validation studies demonstrate a linear calibration model (100-10,000 ng/mL), with dilution integrity verified for the extended range of concentrations experienced in propofol use. Criteria-based validation was achieved, including an average coefficient of variation of 6.5 % and a percent bias of -4.2 ng/mL. The method was evaluated in 12 surgical patients, with monitoring periods lasting up to 30 days following intravenous propofol administrations of 100-3000 mg on the day of surgery. While the concentration ratio of PG to 4-hydroxy propofol metabolite decreased significantly in the days following surgery, PG maintained the highest concentration in all specimens. Both PG and 1-QG were detectable throughout the monitoring periods, including in a patient monitored for 30 days. Lower concentrations were determined for 4-QG and 4-QS, with evidence of detection up to 20 days. Propofol was not detectable in any urine specimens, thereby proving ineffective for identifying drug use. Conclusion: The validated method for quantifying propofol metabolites demonstrates its applicability for the sensitive detection of propofol misuse over a long window of drug-use detection.

The efficacy of ketamine for postoperative pain control in adolescent patients undergoing spinal fusion surgery for idiopathic scoliosis.

The use of ketamine in conjunction with morphine to reduce postoperative pain has been explored in several different surgery subtypes with conflicting results. Ketamine has shown promise to have both opioid sparing and analgesic effects in the postoperative setting. This study aimed to elucidate ketamine's ability to reduce morphine equivalent consumption and improve patient satisfaction after spinal fusion surgery for the correction of idiopathic scoliosis. This surgery is known to be associated with significant postoperative pain which impedes the ability to improve patient satisfaction, and may complicate the recovery timeline. Currently, the standard therapeutic regimen consists of patient-controlled analgesia morphine and the use of other opioids such as hydromorphone. A prospective, randomized double-blinded, placebo-controlled trial was performed to compare the standard morphine equivalent therapy alone against a standard therapy in conjunction with ketamine. Fifty adolescent patients were enrolled and randomized. Results yielded a significant reduction in postoperative morphine equivalent consumption (p = 0.042), adjusted postoperative pain scores (p < 0.001), and incidence of nausea and vomiting (p = 0.045). The application of ketamine as an analgesic in conjunction with the current standard of morphine equivalent therapy may serve as a superior pain control regimen for spinal surgeries in young population. This regimen enhancement may be generalizable to other surgery subtypes within similar populations. LEVEL OF EVIDENCE: Level I.

Analysis of 17 fentanyls in plasma and blood by UPLC-MS/MS with interpretation of findings in surgical and postmortem casework.

The opioid crisis is linked to an increased misuse of fentanyl as well as fentanyl analogs that originate from the illicit drug market. Much of our current understanding of fentanyl and fentanyl analog use in our communities comes from postmortem toxicology findings. In the clinical settings of addiction medicine and pain management, where the opioid abuse potential is high, the use of fentanyl, as well as specific fentanyl analogs, may be underestimated due to limited plasma testing and limited availability of assays with suitable analytical sensitivity and selectivity to detect misuse of fentanyls. We report plasma and blood assays for 17 fentanyls (these include fentanyl, fentanyl analogs, fentanyl metabolites and synthetic precursors) in clinical, and medical examiner, casework. A mixed-mode solid phase extraction of diluted plasma or precipitated blood was optimized for maximum recovery of the fentanyls with minimized matrix effects. Analysis was performed using a Waters ACQUITY UPLC I-Class interfaced with a Waters Xevo TQ-S micro tandem quadrupole mass spectrometer. Method parameters were optimized and validated for precision, accuracy, carryover, linearity and matrix effects. Application studies were performed in postmortem blood obtained in 44 fentanyl-related fatalities and in serial plasma samples from 18 surgical patients receiving intravenous fentanyl therapy while undergoing parathyroidectomy. Fentanyls found in postmortem cases included fentanyl, norfentanyl, despropionyl-fentanyl (4-ANPP), beta-hydroxy fentanyl (ß-OH fentanyl), acetyl fentanyl, acetyl norfentanyl, methoxyacetyl fentanyl, furanyl fentanyl, cyclopropyl fentanyl, and para-fluorobutyryl fentanyl, with fentanyl, norfentanyl, 4-ANPP and ß-OH fentanyl predominating in frequency. Fentanyl concentrations ranged from 0.2 to 56 ng/mL and fentanyl was nearly always found with 4-ANPP, norfentanyl and ß-OH fentanyl. Concentrations of other fentalogs ranged from <1 to 84 ng/mL (extrapolated). In the surgical cases, fentanyl was detected and quantified along with norfentanyl and ß-OH fentanyl, but without detection of 4-ANPP in any of the samples. The association and relative concentrations of ß-OH fentanyl, fentanyl and norfentanyl in the postmortem and clinical studies indicated a metabolic, rather than an illicit, source of ß-OH fentanyl.

Use of jet ventilation in thoracoscopic tracheo-esophageal fistula repair-can both surgeons and anesthesiologists be happy?

Laparoscopic and open thoracic surgery in the neonate typically results in hypercapnea and low cardiac output with often poor surgical visualization as the anesthesiologist attempts to correct the respiratory derangements usually seen. We describe three cases in which jet ventilation provided not only superior ventilation with a return to normocapnea but also ideal operating conditions. In addition, jet ventilation utilizes lower mean airway pressures which typically results in improved cardiac output.

NFATc1 controls skeletal muscle fiber type and is a negative regulator of MyoD activity.

Skeletal muscle comprises a heterogeneous population of fibers with important physiological differences. Fast fibers are glycolytic and fatigue rapidly. Slow fibers utilize oxidative metabolism and are fatigue resistant. Muscle diseases such as sarcopenia and atrophy selectively affect fast fibers, but the molecular mechanisms regulating fiber type-specific gene expression remain incompletely understood. Here, we show that the transcription factor NFATc1 controls fiber type composition and is required for fast-to-slow fiber type switching in response to exercise in vivo. Moreover, MyoD is a crucial transcriptional effector of the fast fiber phenotype, and we show that NFATc1 inhibits MyoD-dependent fast fiber gene promoters by physically interacting with the N-terminal activation domain of MyoD and blocking recruitment of the essential transcriptional coactivator p300. These studies establish a molecular mechanism for fiber type switching through direct inhibition of MyoD to control the opposing roles of MyoD and NFATc1 in fast versus slow fiber phenotypes.

The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10.

Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. Here, we establish a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. We identify a transcriptional enhancer of Mef2c that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. We demonstrate that Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, we show that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes.

Airway responses to desflurane during maintenance of anesthesia and recovery in children with laryngeal mask airways.

BACKGROUND: We sought to characterize the airway responses to desflurane during maintenance of and emergence from anesthesia in children whose airways were supported with laryngeal mask airways (LMAs). METHODS/MATERIALS: Four hundred healthy children were randomized in a 3 : 1 ratio to either desflurane or isoflurane (reference group) during anesthetic maintenance. After induction of anesthesia, anesthesia was maintained with the designated anesthetic. The investigator chose the airway (LMA and facemask), ventilation strategy and when to remove the LMA. The incidence of airway events during maintenance, emergence and recovery was recorded. RESULTS: Ninety percent of children received LMAs. The frequency of major airway events after desflurane (9%) was similar to that after isoflurane (4%) (number needed to harm [NNH] 20), although the frequency of major events after the LMA was removed during deep desflurane anesthesia (15%) was greater than during awake removal (5%) (NNH 10) (P < 0.006) and during deep isoflurane removal (2%) (NNH 8) (P < 0.03). The frequency of airway events of any severity after desflurane was greater than that after isoflurane (39% vs 27%) (P < 0.05). The frequencies of laryngospasm and coughing of any severity after desflurane were greater than those after isoflurane (13% vs 5% and 26% vs 14%, respectively) (P < 0.05). CONCLUSIONS: When an LMA is used during desflurane anesthesia in children, fewer airway events occur when it is removed when the child is awake. Although the time to discharge from recovery was not delayed and no child required overnight admission, caution should be exercised when using an LMA in children who are anesthetized with desflurane.

Alarming levels of carboxyhemoglobin in banked blood.

OBJECTIVE: To determine the level of carboxyhemoglobin found in banked blood in the Albany, NY region. DESIGN: A retrospective descriptive analysis of carboxyhemoglobin (COHb) levels in a series of packed red blood cell (PRBC) units. SETTING: The blood bank of a university tertiary care hospital in Albany, NY. PARTICIPANTS: All PRBC units considered for possible use in pediatric cardiac surgery were first analyzed for levels of COHb. INTERVENTIONS: Only those units with COHb levels of <1.5% were deemed acceptable for use during pediatric cardiac surgery. MEASUREMENTS AND RESULTS: A sample of blood drawn from the sample side arm of each PRBC unit was analyzed on a Chiron 855 Blood Gas Analyzer (Chiron Inc, Emeryville, CA, now Siemens/Bayer RapidLab 865) to determine the level of COHb. The average COHb level was 0.78% (standard deviation +/- 1.48%), and out of the 468 units tested, 48 (10.3%) had COHb levels of 1.5% or greater. The highest recorded COHb level was 12%. CONCLUSIONS: The transfusion of PRBC units may artificially elevate readings of COHb and cause confusion over possible causes. Certain high-risk populations (eg, cyanotic neonates undergoing cardiopulmonary bypass) may be especially at risk. Although levels of COHb in the US blood supply are dropping, institutions may want to consider analyzing COHb levels in their PRBC units before transfusion in these high-risk populations.

Neuronal activity regulates phosphorylation-dependent surface delivery of G protein-activated inwardly rectifying potassium channels.

G protein-activated inwardly rectifying K(+) (GIRK) channels regulate neuronal excitability by mediating inhibitory effects of G protein-coupled receptors for neurotransmitters and neuromodulators. Notwithstanding many studies reporting modulation of GIRK channel function, whether neuronal activity regulates GIRK channel trafficking remains an open question. Here we report that NMDA receptor activation in cultured dissociated hippocampal neurons elevates surface expression of the GIRK channel subunits GIRK1 and GIRK2 in the soma, dendrites, and dendritic spines within 15 min. This activity-induced increase in GIRK surface expression requires protein phosphatase-1-mediated dephosphorylation of a serine residue (Ser-9) preceding the GIRK2 Val-13/Leu-14 (VL) internalization motif, thereby promoting channel recycling. Because activation of GIRK channels hyperpolarizes neuronal membranes, the NMDA receptor-induced regulation of GIRK channel trafficking may represent a dynamic adjustment of neuronal excitability in response to inhibitory neurotransmitters and/or neuromodulators.

Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors.

Vascular development begins when mesodermal cells differentiate into endothelial cells, which then form primitive vessels. It has been hypothesized that endothelial-specific gene expression may be regulated combinatorially, but the transcriptional mechanisms governing specificity in vascular gene expression remain incompletely understood. Here, we identify a 44 bp transcriptional enhancer that is sufficient to direct expression specifically and exclusively to the developing vascular endothelium. This enhancer is regulated by a composite cis-acting element, the FOX:ETS motif, which is bound and synergistically activated by Forkhead and Ets transcription factors. We demonstrate that coexpression of the Forkhead protein FoxC2 and the Ets protein Etv2 induces ectopic expression of vascular genes in Xenopus embryos, and that combinatorial knockdown of the orthologous genes in zebrafish embryos disrupts vascular development. Finally, we show that FOX:ETS motifs are present in many known endothelial-specific enhancers and that this motif is an efficient predictor of endothelial enhancers in the human genome.

Modulation of basal and receptor-induced GIRK potassium channel activity and neuronal excitability by the mammalian PINS homolog LGN.

G protein-activated inwardly rectifying potassium (GIRK) channels mediate slow synaptic inhibition and control neuronal excitability. It is unknown whether GIRK channels are subject to regulation by guanine dissociation inhibitor (GDI) proteins like LGN, a mammalian homolog of Drosophila Partner of Inscuteable (mPINS). Here we report that LGN increases basal GIRK current but reduces GIRK activation by metabotropic transmitter receptors coupled to Gi or Go, but not Gs. Moreover, expression of its N-terminal, TPR-containing protein interaction domains mimics the effects of LGN in mammalian cells, probably by releasing sequestered endogenous LGN. In hippocampal neurons, expression of LGN, or LGN fragments that mimic or enhance LGN activity, hyperpolarizes the resting potential due to increased basal GIRK activity and reduces excitability. Using Lenti virus for LGN RNAi to reduce endogenous LGN levels in hippocampal neurons, we further show an essential role of LGN for maintaining basal GIRK channel activity and for harnessing neuronal excitability.

Severe polycythemia in an infant with uncorrected tetralogy of Fallot presenting for noncardiac surgery.

Pediatric patients with uncorrected cyanotic congenital heart diseases may present for noncardiac surgery. Associated congenital defects and severe uncompensated secondary erythrocytosis may complicate their anesthetic management. We describe the uncomplicated anesthetic for open G-tube placement of an ex-premature 8-month-old infant with uncorrected tetralogy of Fallot, multiple associated congenital anomalies, and a preoperative hematocrit of 78% and review the anesthetic implications of severe polycythemia.

Alarming levels of carboxyhemoglobin in a unit of banked blood.

IMPLICATIONS: Increased levels of carboxyhemoglobin (COHb) are frequently found in units of packed red blood cells. We report a congenital heart surgery where increased levels of COHb were found in the patient after a blood transfusion and hypothesize that this phenomenon could be dangerous in a cyanotic newborn undergoing open heart surgery.