Functional analysis of RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia.

BACKGROUND: A major bottleneck to the introduction of noninvasive presymptomatic diagnostic tests for the pharmacogenetic disorder malignant hyperthermia is the lack of functional data for associated variants. METHODS: We screened 50 genes having a potential role in skeletal muscle calcium homeostasis using the HaloPlex™ (Agilent Technologies, Santa Clara, CA, USA) target enrichment system and next-generation sequencing. Twenty-one patients with a history of a clinical malignant hyperthermia reaction together with a positive in vitro contracture test were included. Eight variants in RYR1 were subsequently introduced into the cDNA for the human ryanodine receptor gene and tested in cultured human embryonic kidney (HEK293) cells for their effect on calcium release from intracellular stores in response to the ryanodine receptor-1 agonist 4-chloro-m-cresol using fura-2 as calcium indicator. Each variant was subjected to in silico curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 variant curation expert panel guidelines. RESULTS: Potentially causative RYR1 variants were identified in 15 patients. Of these, two families carried two RYR1 variants, five variants had been previously reported as 'pathogenic', two variants had been previously reported as 'likely benign', and eight were of 'uncertain significance'. Of these eight variants, four showed hypersensitivity to 4-chloro-m-cresol. Three variants were reclassified as either 'pathogenic' or 'likely pathogenic'. Two were classified as 'benign', whilst three remained of 'uncertain significance'. CONCLUSIONS: Three (p.Tyr1711Cys, p.Val2280Ile, and p.Arg4737Gln) additional variants can be added to the list of RYR1 disease-associated variants managed by the European Malignant Hyperthermia Group. These can therefore be used diagnostically in the future. Three variants (p.Glu2348Gly, p.Asn2634Lys, and p.Arg3629Trp) that remained classified as of uncertain significance require further family studies or a different functional test to determine clinical relevance in malignant hyperthermia.

Levobupivacaine plasma concentrations following repeat caudal anesthetics.

AIM: A single caudal anesthetic at the start of lower abdominal surgery is unlikely to provide prolonged analgesia. A second caudal at the end of the procedure extends the analgesia duration but total plasma concentrations may be associated with toxicity. Our aim was to measure total plasma levobupivacaine concentrations after repeat caudal anesthesia in infants and to generate a pharmacokinetic model for prediction of plasma concentrations after repeat caudal anesthesia in neonates, infants and children. METHODS: Infants undergoing definitive repair of anorectal malformations or Hirschsprung's disease received a second caudal anesthesia at the end of the procedure. Total levobupivacaine concentrations were assayed 3-4 times in the first 6 h after the initial caudal. These data were pooled with data from four studies describing plasma concentrations after levobupivacaine caudal or spinal anesthesia. Population pharmacokinetic parameters were estimated using nonlinear mixed-effects models. Covariates included postmenstrual age and body weight. Parameter estimates were used to simulate concentrations after a repeat levobupivacaine 2.5 mg kg-1 caudal at 3 or 4 h following an initial levobupivacaine 2.5 mg kg-1 caudal. RESULTS: Twenty-one infants (postnatal age 11-32 weeks, gestation 37-39 weeks, weight 5.2-8.6 kg) were included. The measured peak plasma concentration after repeat caudal levobupivacaine 2.5 mg kg-1 4 h after initial caudal was 1.38 mg L-1 (95% prediction interval 0.60-2.6 mg L-1 ) and 3 h after initial caudal was 1.46 mg L-1 (0.60-2.80) mg L-1 . Simulation of total plasma concentrations in neonates (7 kg, 57 weeks postmenstrual age) given caudal levobupivacaine 4 h after the initial caudal were 1.76 mg L-1 (0.68-3.50) mg L-1 if 2.5 mg kg-1 levobupivacaine was used and 0.88 mg L-1 (0.34-1.73) mg L-1 if 1.25 mg kg-1 of 0.125% levobupivacaine was used. In simulated older children (20 kg, 6 years), the mean maximum concentration was 1.43 mg L-1 (0.60-2.70) mg L-1 if 2.5 mg kg-1 levobupivacaine was repeated at 3 h. CONCLUSION: Repeat caudal levobupivacaine 2.5 mg kg-1 at 3 h after an initial 2.5 mg kg-1 dose does not exceed the concentration associated with systemic local anesthetic toxicity. In 2.5% of simulated neonates (weight 3.8 kg, PMA 40 weeks), repeat caudal anesthesia demonstrates broaching of the lower concentration limit associated with toxicity at both 3 and 4 h after initial caudal.

Pre-operative exercise and pyrexia as modifying factors in malignant hyperthermia (MH).

Malignant hyperthermia (MH) is a life-threatening reaction triggered by volatile anesthetics and succinylcholine. MH is caused by mutations in the skeletal muscle ryanodine receptor (RYR1) gene, as is rhabdomyolysis triggered by exertion and/or pyrexia. The discrepancy between the prevalence of risk genotypes and actual MH incidence remains unexplained. We investigated the role of pre-operative exercise and pyrexia as potential MH modifying factors. We included cases from 5 MH referral centers with 1) clinical features suggestive of MH, 2) confirmation of MH susceptibility on Contracture Testing (IVCT or CHCT) and/or RYR1 genetic testing, and a history of 3) strenuous exercise within 72 h and/or pyrexia >37.5 °C prior to the triggering anesthetic. Characteristics of MH-triggering agents, surgery and succinylcholine use were collected. We identified 41 cases with general anesthesias resulting in an MH event (GA+MH, n = 41) within 72 h of strenuous exercise and/or pyrexia. We also identified previous general anesthesias without MH events (GA-MH, n = 51) in the index cases and their MH susceptible relatives. Apart from pre-operative exercise and/or pyrexia, trauma and acute abdomen as surgery indications, emergency surgery and succinylcholine use were also more common with GA+MH events. These observations suggest a link between pre-operative exercise, pyrexia and MH.

Junctional membrane Ca2+ dynamics in human muscle fibers are altered by malignant hyperthermia causative RyR mutation.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca2+-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca2+-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca2+ sensor of Ca2+ dynamics in the junctional space. The sensor was used to assess the basal Ca2+-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca2+ handling under defined ionic conditions.

Self-Organized Nanostructure Modified Microelectrode for Sensitive Electrochemical Glutamate Detection in Stem Cells-Derived Brain Organoids.

Neurons release neurotransmitters such as glutamate to communicate with each other and to coordinate brain functioning. As increased glutamate release is indicative of neuronal maturation and activity, a system that can measure glutamate levels over time within the same tissue and/or culture system is highly advantageous for neurodevelopmental investigation. To address such challenges, we develop for the first time a convenient method to realize functionalized borosilicate glass capillaries with nanostructured texture as an electrochemical biosensor to detect glutamate release from cerebral organoids generated from human embryonic stem cells (hESC) that mimic various brain regions. The biosensor shows a clear catalytic activity toward the oxidation of glutamate with a sensitivity of 93 ± 9.5 nA·µM-1·cm-2. It was found that the enzyme-modified microelectrodes can detect glutamate in a wide linear range from 5 µM to 0.5 mM with a limit of detection (LOD) down to 5.6 ± 0.2 µM. Measurements were performed within the organoids at different time points and consistent results were obtained. This data demonstrates the reliability of the biosensor as well as its usefulness in measuring glutamate levels across time within the same culture system.

Generation of Adrenal Chromaffin-like Cells from Human Pluripotent Stem Cells.

Adrenomedullary chromaffin cells are catecholamine (CA)-producing cells originating from trunk neural crest (NC) via sympathoadrenal progenitors (SAPs). We generated NC and SAPs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro via BMP2/FGF2 exposure, ascertained by qPCR and immunoexpression of SOX10, ASCL1, TFAP2α, and PHOX2B, and by fluorescence-activated cell sorting selection for p75NTR and GD2, and confirmed their trunk-like HOX gene expression. We showed that continuing BMP4 and curtailing FGF2 in vitro, augmented with corticosteroid mimetic, induced these cells to upregulate the chromaffin cell-specific marker PNMT and other CA synthesis and storage markers, and we demonstrated noradrenaline and adrenaline by Faglu and high-performance liquid chromatography. We showed these human cells' SAP-like property of migration and differentiation into cells expressing chromaffin cell markers by implanting them into avian embryos in vivo and in chorio-allantoic membrane grafts. These cells have the potential for investigating differentiation of human chromaffin cells and for modeling diseases involving this cell type.

Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia.

BACKGROUND: Central core disease and malignant hyperthermia are human disorders of skeletal muscle resulting from aberrant Ca2+ handling. Most malignant hyperthermia and central core disease cases are associated with amino acid changes in the type 1 ryanodine receptor (RyR1), the skeletal muscle Ca2+-release channel. Malignant hyperthermia exhibits a gain-of-function phenotype, and central core disease results from loss of channel function. For a variant to be classified as pathogenic, functional studies must demonstrate a correlation with the pathophysiology of malignant hyperthermia or central core disease. OBJECTIVE: We assessed the pathogenicity of four C-terminal variants of the ryanodine receptor using functional analysis. The variants were identified in families affected by either malignant hyperthermia or central core disease. METHODS: Four variants were introduced separately into human cDNA encoding the skeletal muscle ryanodine receptor. Following transient expression in HEK-293T cells, functional studies were carried out using calcium release assays in response to an agonist. Two previously characterized variants and wild-type skeletal muscle ryanodine receptor were used as controls. RESULTS: The p.Met4640Ile variant associated with central core disease showed no difference in calcium release compared to wild-type. The p.Val4849Ile variant associated with malignant hyperthermia was more sensitive to agonist than wild-type but did not reach statistical significance and two variants (p.Phe4857Ser and p.Asp4918Asn) associated with central core disease were completely inactive. CONCLUSIONS: The p.Val4849Ile variant should be considered a risk factor for malignant hyperthermia, while the p.Phe4857Ser and p.Asp4918Asn variants should be classified as pathogenic for central core disease.

Comparative Plasma and Cerebrospinal Fluid Pharmacokinetics of Paracetamol After Intravenous and Oral Administration.

BACKGROUND: We compared plasma and cerebrospinal fluid (CSF) pharmacokinetics of paracetamol after intravenous (IV) and oral administration to determine dosing regimens that optimize CSF concentrations. METHODS: Twenty-one adult patients were assigned randomly to 1 g IV, 1 g oral or 1.5 g oral paracetamol. An IV cannula and lumbar intrathecal catheter were used to sample venous blood and CSF, respectively, over 6 hours. The plasma and CSF maximum concentrations (Cmax), times to maximum concentrations (Tmax), and area under the plasma and CSF concentration-time curves (AUCs) were calculated using noncompartmental techniques. Significance was defined by P < .0167 (Bonferroni correction for 3 comparisons for each parameter). Probability (X < Y) (p″) with Bonferroni corrected 95% confidence intervals (CIs) were calculated (CIs including 0.5 meet the null hypothesis). Results are presented as median (range) or p″ (CI). P values are listed as 1 g IV vs 1 g orally, 1 g IV vs 1.5 g orally and 1 g orally vs 1.5 g orally, respectively. RESULTS: Wide variation in measured paracetamol concentrations was observed, especially in the oral groups. The median plasma Cmax in the 1 g IV group was significantly greater than the oral groups. In contrast, the median CSF Cmax was not different between groups. The median plasma Tmax in the 1 g IV group was 105 and 75 minutes earlier than in the 1 and 1.5 g oral groups. The median CSF Tmax was not significantly different between groups. The median plasma AUC (total) was not significantly different between groups; however, in the first hour, the median plasma AUC was significantly greater in the IV group than in the oral groups. In the second hour, there was no difference between groups. The median CSF AUC (total) did not significantly differ between groups; however, in the first hour, the median CSF AUC was significantly greater in the IV compared with the orally groups. In the second hour, there was no difference between groups. Our analysis indicated that the median Cmax, Tmax, and AUC values lacked precision because of small sample sizes. CONCLUSIONS: Peak plasma concentrations were greater and reached earlier after IV than oral dosing. Evidence for differences in CSF Cmax and Tmax was lacking because of the small size of this study.

Peritumoural glutamate correlates with post-operative seizures in supratentorial gliomas.

To examine the impact of glutamate on post-operative seizures and survival in a cohort of patients with grade II to IV supratentorial glioma. A retrospective analysis was performed on 216 patients who underwent surgery for supratentorial gliomas. Primary explanatory variables were peritumoural and/or tumoural glutamate concentrations, glutamate transporter expression (EAAT2 and SXC). Univariate and multivariate survival analysis was performed with primary outcomes of time to first post-operative seizure and overall survival. Subgroup analysis was performed in patients with de novo glioblastomas who received adjuvant chemoradiotherapy. 47 (21.8 %), 34 (15.8 %) and 135 (62.5 %) WHO grade II, III and IV gliomas respectively were followed for a median of 15.8 months. Following multivariate analysis, there was a non-significant association between higher peritumoural glutamate concentrations and time to first post-operative seizure (HR 2.07, CI 0.98-4.37, p = 0.06). In subgroup analysis of 81 glioblastoma patients who received adjunct chemoradiotherapy, peritumoural glutamate concentration was significantly associated with time to first post-operative seizure (HR 3.10, CI 1.20-7.97, p = 0.02). In both the overall cohort and subgroup analysis no glutamate cycle biomarkers were predictive of overall survival. Increased concentrations of peritumoural glutamate were significantly associated with shorter periods of post-operative seizure freedom in patients with de novo glioblastomas treated with adjuvant chemoradiotherapy. No glutamate cycle biomarkers were predictive of overall survival. These results suggest that therapies targeting glutamate may be beneficial in tumour associated epilepsy.

Pharmacokinetics of levobupivacaine following infant spinal anesthesia.

BACKGROUND: Infant spinal anesthesia with levobupivacaine has been promoted as a technique to reduce both the risk of postoperative apnea and exposure to volatile anesthesia. There is, however, no pharmacokinetic data to support the currently recommended doses. AIMS: Our aim was to determine whether infant levobupivacaine spinal anesthesia is associated with plasma concentrations consistent with a low risk of local anesthetic systemic toxicity. METHODS: This was an open-label pharmacokinetic safety and tolerability study of levobupivacaine spinal anesthesia in infants <55 weeks Post Menstrual Age undergoing lower abdominal surgery. Infants received a spinal anesthetic with levobupivacaine 1 mg·kg(-1) in the left lateral position. RESULTS: Spinal anesthesia was successful in 25 (86.2%) of 29 infants (postmenstrual age 36-52 weeks; weight 2.2-4.7 kg). The median (IQR) total venous levobupivacaine plasma concentrations was 0.33 (0.25-0.42) µg·ml(-1) and unbound venous levobupivacaine was 19.5 (14.5-38) ng·ml(-1) . Median protein binding was 93.5 (91.4-96%). Alpha-1 acid glycoprotein concentrations were 0.25 (0.17-0.37) g·l(-1) and albumin concentrations were 29 (24-32) g·l(-1) . CONCLUSION: Total plasma concentrations and unbound (free) concentration of levobupivacaine were consistently lower than concentrations reported in cases of pediatric local anesthetic toxicity. In a small number of infants requiring a repeat spinal of 1 mg·kg(-1) was also associated with acceptable total and free concentrations. We conclude that levobupivacaine at 1 mg·kg(-1) is associated with no systemic side effects in infants receiving awake spinal anesthesia.

The peroxisome proliferator activated receptor gamma agonist pioglitazone increases functional expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in human glioblastoma cells.

Glioma cells release glutamate through expression of system xc-, which exchanges intracellular glutamate for extracellular cysteine. Lack of the excitatory amino acid transporter 2 (EAAT2) expression maintains high extracellular glutamate levels in the glioma microenvironment, causing excitotoxicity to surrounding parenchyma. Not only does this contribute to the survival and proliferation of glioma cells, but is involved in the pathophysiology of tumour-associated epilepsy (TAE). We investigated the role of the peroxisome proliferator activated receptor gamma (PPARγ) agonist pioglitazone in modulating EAAT2 expression in glioma cells. We found that EAAT2 expression was increased in a dose dependent manner in both U87MG and U251MG glioma cells. Extracellular glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in both U87MG and U251MG cells at a concentration of ≥ 30 µM pioglitazone (p < 0.05). The PPARγ antagonist GW9662 inhibited the effect of pioglitazone on extracellular glutamate levels, indicating PPARγ dependence. In addition, pioglitazone significantly reduced cell viability of U87MG and U251MG cells at ≥ 30 µM and 100 µM (p < 0.05) respectively. GW9662 also significantly reduced viability of U87MG and U251MG cells with 10 µM and 30 µM (p < 0.05) respectively. The effect on viability was partially dependent on PPARγ activation in U87MG cells but not U251MG cells, whereby PPARγ blockade with GW9662 had a synergistic effect. We conclude that PPARγ agonists may be therapeutically beneficial in the treatment of gliomas and furthermore suggest a novel role for these agents in the treatment of tumour associated seizures through the reduction in extracellular glutamate.

Absorption characteristics of epidural levobupivacaine with adrenaline and clonidine in children.

AIM: To determine if the addition of adrenaline, clonidine, or their combination altered the pharmacokinetic profile of levobupivacaine administered via the caudal epidural route in children. METHODS: Children aged <18 years old scheduled to undergo sub-umbilical surgery were administered caudal levobupivacaine plain 2.5 mg · ml(-1) or with adjuvants adrenaline 5 mcg · ml(-1) or clonidine 2 mcg · ml(-1) or their combination. Covariate analysis included weight and postnatal age (PNA). Time-concentration profile analysis was undertaken using nonlinear mixed effects models. A one-compartment linear disposition model with first-order input and first-order elimination was used to describe the data. The effect of either clonidine or adrenaline on absorption was investigated using a scaling parameter (Fabs(CLON), Fabs(ADR)) applied to the absorption half-life (Tabs). RESULTS: There were 240 children (median weight 11.0, range 1.9-56.1 kg; median postnatal age 16.7, range 0.6-167.6 months). Absorption of levobupivacaine was faster when mixed with clonidine (Fabs(CLON) 0.60; 95%CI 0.44, 0.83) but slower when mixed with adrenaline (Fabs(ADR) 2.12; 95%CI 1.45, 3.08). The addition of adrenaline to levobupivacaine resulted in a bifid absorption pattern. While initial absorption was unchanged (Tabs 0.15 h 95%CI 0.12, 0.18 h), there was a late absorption peak characterized by a Tabs(LATE) 2.34 h (95%CI 1.44, 4.97 h). The additional use of clonidine with adrenaline had minimal effect on the bifid absorption profile observed with adrenaline alone. Neither clonidine nor adrenaline had any effect on clearance. The population parameter estimate for volume of distribution was 157 l 70 kg(-1). Clearance was 6.5 l · h(-1) 70 kg(-1) at 1-month PNA and increased with a maturation half-time of 1.6 months to reach 90% of the mature value (18.5 l · h(-1) 70 kg(-1)) by 5 months PNA. CONCLUSIONS: The addition of adrenaline decreases the rate of levobupivacaine systemic absorption, reducing peak concentration by half. Levobupivacaine concentrations with adrenaline adjuvant were reduced compared to plain levobupivacaine for up to 3.5 hours. Clonidine as an adjuvant results in faster systemic absorption of levobupivacaine and similar concentration time profile to levobupivacaine alone. Adding adrenaline with clonidine does not alter the concentration profile observed with adrenaline alone.

Glutamate is associated with a higher risk of seizures in patients with gliomas.

OBJECTIVE: To investigate the relationship of glutamate and glutamate transporter expression in human gliomas and surrounding peritumoral brain to the presence of tumor-associated seizures (TAS). METHODS: We studied a retrospective (group 1: 190 patients) and then a prospective (group 2: 98 patients) cohort of patients who underwent a craniotomy for a supratentorial glioma. Tumor and peritumor tissue specimens were assayed for glutamate concentration and expression of glial glutamate transporters. Differences between the seizure (TAS) and seizure-free (non-TAS) groups were compared. RESULTS: A total of 42% of patients had TAS, with 95% of seizures first occurring preoperatively. Clinical factors independently associated with risk of TAS were younger age, temporal lobe location, and tumors with oligodendroglial components. Molecular features in tumor specimens associated with TAS were higher glutamate concentrations, reduced EAAT2 expression, and increased system X(c)(-) expression. In group 2, these results were also replicated in the peritumor tissue. Logistic regression analysis identified raised glutamate concentrations in tumor and peritumor tissue, increased expression of peritumor system X(c)(-), younger age, temporal lobe location, and tumors with oligodendroglial components as independently predictive of preoperative seizures. CONCLUSION: Relative increased glutamate concentration in gliomas, and altered glutamate transporter expression, are associated with the presence of TAS and may play a mechanistic role in their pathogenesis.

Automated responsiveness monitor to titrate propofol sedation.

BACKGROUND: In previous studies, we showed that failure to respond to automated responsiveness monitor (ARM) precedes potentially serious sedation-related adversities associated with loss of responsiveness, and that the ARM was not susceptible to false-positive responses. It remains unknown, however, whether loss and return of response to the ARM occur at similar sedation levels. We hypothesized that loss and return of response to the ARM occur at similar sedation levels in individual subjects, independent of the propofol effect titration scheme. METHODS: Twenty-one healthy volunteers aged 20-45 yr underwent propofol sedation using an effect-site target-controlled infusion system and two different dosing protocol schemes. In all, we increased propofol effect-site concentration (Ce) until loss of response to the ARM occurred. Subsequently, the propofol Ce was decreased either by a fixed percentage (20%, 30%, 40%, 50%, 60%, and 70%; fixed percentage protocol, n = 10) or by a linear deramping (0.1, 0.2, and 0.3 microg x mL(-1) x min(-1); deramping protocol, n = 11) until the ARM response returned. Consequently, the propofol Ce was maintained at the new target for a 6-min interval (Ce plateau) during which arterial samples for propofol determination were obtained, and a clinical assessment of sedation (Observer's Assessment of Alertness/Sedation [OAA/S] score) performed. Each participant in the two protocols experienced each percentage or deramping rate of Ce decrease in random order. The assumption of steady state was tested by plotting the limits of agreement between the starting and ending plasma concentration (Cp) at each Ce plateau. The probability of response to the ARM as a function of propofol Ce, Bispectral Index (BIS) of the electroencephalogram, and OAA/S score was estimated, whereas the effect of the protocol type on these estimates was evaluated using the nested model approach (NONMEM). The combined effect of propofol Ce and BIS on the probability for ARM response was also evaluated using a fractional probability model (P(BIS/Ce)). RESULTS: The measured propofol Cp at the beginning and the end of the Ce plateau was almost identical. The Ce50 of propofol for responding to the ARM was 1.73 (95% confidence interval: 1.55-2.10) microg/mL, whereas the corresponding BIS50 was 75 (71.3-77). The OAA/S50 probability for ARM response was 12.5/20 (12-13.4). A fractional probability (P(BIS/Ce)) model for the combined effect of BIS and Ce fitted the data best, with an estimated contribution for BIS of 63%. Loss and return of ARM response occurred at similar sedation levels in individual subjects. CONCLUSIONS: Reproducible ARM dynamics in individual subjects compares favorably with clinical and electroencephalogram sedation end points and suggests that the ARM could be used as an independent instrumental guide of drug effect during propofol-only sedation.

N-acetylcysteine attenuates the decline in muscle Na+,K+-pump activity and delays fatigue during prolonged exercise in humans.

Reactive oxygen species (ROS) have been linked with both depressed Na(+),K(+)-pump activity and skeletal muscle fatigue. This study investigated N-acetylcysteine (NAC) effects on muscle Na(+),K(+)-pump activity and potassium (K(+)) regulation during prolonged, submaximal endurance exercise. Eight well-trained subjects participated in a double-blind, randomised, crossover design, receiving either NAC or saline (CON) intravenous infusion at 125 mg kg(-1) h(-1) for 15 min, then 25 mg kg(-1) h(-1) for 20 min prior to and throughout exercise. Subjects cycled for 45 min at 71% , then continued at 92% until fatigue. Vastus lateralis muscle biopsies were taken before exercise, at 45 min and fatigue and analysed for maximal in vitro Na(+),K(+)-pump activity (K(+)-stimulated 3-O-methyfluorescein phosphatase; 3-O-MFPase). Arterialized venous blood was sampled throughout exercise and analysed for plasma K(+) and other electrolytes. Time to fatigue at 92% was reproducible in preliminary trials (c.v. 5.6 +/- 0.6%) and was prolonged with NAC by 23.8 +/- 8.3% (NAC 6.3 +/- 0.5 versus CON 5.2 +/- 0.6 min, P < 0.05). Maximal 3-O-MFPase activity decreased from rest by 21.6 +/- 2.8% at 45 min and by 23.9 +/- 2.3% at fatigue (P < 0.05). NAC attenuated the percentage decline in maximal 3-O-MFPase activity (%Deltaactivity) at 45 min (P < 0.05) but not at fatigue. When expressed relative to work done, the %Deltaactivity-to-work ratio was attenuated by NAC at 45 min and fatigue (P < 0.005). The rise in plasma [K(+)] during exercise and the Delta[K(+)]-to-work ratio at fatigue were attenuated by NAC (P < 0.05). These results confirm that the antioxidant NAC attenuates muscle fatigue, in part via improved K(+) regulation, and point to a role for ROS in muscle fatigue.

Nitrous oxide and anesthetic requirement for loss of response to command during propofol anesthesia.

The blood concentration associated with loss of response (LOR) to command in 50% of subjects (CP50(LOR)) is an important measure of anesthetic potency. We therefore determined the CP50(LOR) in 40 healthy surgical patients, aged 18-60 yr old, receiving propofol alone or propofol with 67% nitrous oxide (N2O). Patients were randomized to receive 100% oxygen or 67% N2O in oxygen via facemask. Three minutes later, a target-controlled propofol infusion was commenced at a concentration determined by the response of the previous patient in the same group. Fifteen minutes later, response to command was assessed by a blinded observer. Arterial blood samples were taken for propofol assay, and the bispectral index (BIS) was monitored continuously. At testing for response to command, both the measured and target propofol concentrations were significantly larger and BIS values significantly smaller in the propofol-alone group compared with the propofol-N2O group. The CP50(LOR) of propofol in the propofol-alone group was 4.58 mug/mL (95% confidence interval [CI], 1.14-15.36) and 2.67 microg/mL (95% CI, 2.28-3.17) in the propofol-N2O group. The BIS value when 50% of patients responded to command was 60 (95% CI, 55-65) in the propofol-alone group and 75 (95% CI, 73-83) in the propofol-N2O group.

Pharmacokinetics of intravenous N-acetylcysteine in men at rest and during exercise.

OBJECTIVE: We aimed to determine the pharmacokinetics (PK) of N-acetylcysteine (NAC) at rest and during exercise when given by continuous intravenous infusion intended to maintain relatively constant plasma concentrations. METHODS: Plasma concentrations of NAC were measured in 24 healthy male subjects during and after a two-stage intravenous infusion designed to provide constant NAC concentrations during cycling exercise, including intense exercise to fatigue. RESULTS: A three-compartment, open PK model was the best fit using population PK analysis with NONMEM. Whole-body clearance (CL) was 0.58 l kg(-1) h(-1) (95% CI 0.44-0.72) for reduced NAC (NACR) and 0.16 (0.13-0.20) l kg(-1) h(-1) for total NAC (NACT). The central volume of distribution (V1) was 0.064 (0.008-0.12) l kg(-1) for NACR and 0.037 (0.02-0.06) l kg(-1) for NACT. Exercise was a significant covariate in the model, resulting in a 25 and 23% reduction in CL of NACR and NACT, respectively. V1 in our subjects was smaller than expected, resulting in higher-than-anticipated initial concentrations of NAC. Despite these findings, the incidence of adverse effects attributable to NAC was minimal without using prophylactic or concomitant drug therapy. CONCLUSIONS: NAC can be given to healthy exercising men by intravenous infusion and to the plasma concentrations seen in this study with minimal adverse effects due to the drug. The PK parameters of NAC at rest in volunteers are consistent with previously reported values and are significantly altered by vigorous cycling exercise.

Induction speed is not a determinant of propofol pharmacodynamics.

BACKGROUND: Evidence suggests that the rate at which intravenous anesthetics are infused may influence their plasma-effect site equilibration. The authors used five different rates of propofol administration to test the hypothesis that different sedation endpoints occur at the same effect site propofol concentration, independent of the infusion rate. The authors concurrently evaluated the automated responsiveness monitor (ARM) against other sedation measures and the propofol effect site concentration. METHODS: With Human Studies Committee approval, 18 healthy volunteers received five consecutive target-controlled propofol infusions. During each infusion, the effect site concentration was increased by a rate of 0.1, 0.3, 0.5, 0.7, or 0.9 microg . ml . min. The Bispectral Index and ARM were recorded at frequent intervals. The times of syringe drop and loss and recovery of responsiveness were noted. Pharmacokinetic and pharmacodynamic modeling was performed using NONMEM. RESULTS: When the correct rate of plasma-effect site equilibration was determined for each individual (plasma-effect site equilibration = 0.17 min, time to peak effect = 2.7 min), the effect site concentrations associated with each clinical measure were not affected by the rate of increase of effect site propofol concentration. ARM correlated with all clinical measures of drug effect. Subjects invariably stopped responding to ARM at lower effect site propofol concentrations than those associated with loss of responsiveness. CONCLUSIONS: : Population-based pharmacokinetics, combined with real-time electroencephalographic measures of drug effect, may provide a means to individualize pharmacodynamic modeling during target-controlled drug delivery. ARM seems useful as an automated measure of sedation and may provide the basis for automated monitoring and titration of sedation for a propofol delivery system.

Latency of pupillary reflex dilation during general anesthesia.

Areas of insensibility produced by neuraxial anesthesia or peripheral nerve blocks can be detected during general anesthesia by failure of noxious stimulation to trigger pupillary reflex dilation. We examined the latency of pupillary reflex dilation and the effect of fentanyl on the latency of reflex dilation during anesthesia in nine volunteers. We hypothesized that the reflex was generated by slowly conducting C nociceptive fibers and would be significantly delayed if a distal dermatome (L(4)) was stimulated compared with a proximal dermatome (C(5)). We also hypothesized that fentanyl would prolong the latency and alter the shape of the reflex. After induction of general anesthesia, pupillary reflex dilation was measured with an infrared pupillometer every 5 min after stimulations of the L(4) and C(5) dermatomes. Fentanyl (3 microg/kg) was then given intravenously. Pupillary reflex dilation latencies were calculated by examining each individual measurement. After 3 h, naloxone (400 microg) was given intravenously; anesthesia was then discontinued. Pupillary reflex dilation had a long latency and consisted of distinct early and late phases. No differences were found between latencies of reflex dilation after simulation of L(4) and C(5) dermatomes either before or after fentanyl administration. Fentanyl at high concentrations essentially eliminated pupillary reflex dilation; but over the 180-min observation period, first early and then late dilation returned. Fentanyl produced a small increase in the latency of the initial early dilation. We conclude that pupillary reflex dilation during anesthesia is not initiated by slowly conducting C fibers and that fentanyl depresses the reflex in a stereotypical manner.

Dantrolene reduces the threshold and gain for shivering.

UNLABELLED: Dantrolene is used for treatment of life-threatening hyperthermia, yet its thermoregulatory effects are unknown. We tested the hypothesis that dantrolene reduces the threshold (triggering core temperature) and gain (incremental increase) of shivering. Healthy volunteers were evaluated on 2 random days: control and dantrolene (approximately 2.5 mg/kg plus a continuous infusion). In Study 1, 9 men were warmed until sweating was provoked and then cooled until arteriovenous shunt constriction and shivering occurred. Sweating was quantified on the chest using a ventilated capsule. Absolute right middle fingertip blood flow was quantified using venous-occlusion volume plethysmography. A sustained increase in oxygen consumption identified the shivering threshold. In Study 2, 9 men were given cold lactated Ringer's solution i.v. to reduce core temperature approximately 2 degrees C/h. Cooling was stopped when shivering intensity no longer increased with further core cooling. The gain of shivering was the slope of oxygen consumption versus core temperature regression. In Study 1, sweating and vasoconstriction thresholds were similar on both days. In contrast, shivering threshold decreased 0.3 +/- 0.3 degrees C, P = 0.004, on the dantrolene day. In Study 2, dantrolene decreased the shivering threshold from 36.7 +/- 0.2 to 36.3 +/- 0.3 degrees C, P = 0.01 and systemic gain from 353 +/- 144 to 211 +/- 93 mL.min(-1).degrees C(-1), P = 0.02. Thus, dantrolene substantially decreased the gain of shivering, but produced little central thermoregulatory inhibition. IMPLICATIONS: Dantrolene substantially decreases the gain of shivering but produces relatively little central thermoregulatory inhibition. It thus seems unlikely to prove more effective than conventional muscle relaxants for treatment of life-threatening hyperthermia.