Ecofriendly Long Life Nanocomposite Sensors for Determination of Carbachol in Presence of Choline: Application in Ophthalmic Solutions and Biological Fluids.

Several emerging nano scale forms of carbon are showing great promise in electrochemical sensing such as graphene and multi-walled carbon nanotubes (MWCNTs). Herein we present an ecofriendly method to fabricate long life and sensitive ion selective sensors based on graphene and MWCNTs nanocomposites with no need for volatile organic solvents. Both sensors were fabricated, for the analysis of carbachol in ophthalmic solutions, plasma and urine where ion- association complex was formed between cationic carbachol and anionic Sodium tetra phenyl borate (NaTBP) in a ratio 1:1. Both sensors were evaluated according to the IUPAC recommendation data, revealing linear response in the concentration range 10-7 M to 10-2 M with near Nernstian slopes 50.80 ± 5 and 58.14 ± 3 mV/decade and correlation coefficients 0.9992 and 0.9998 for graphene and MWCNTs based sensors, respectively. Both sensors were successfully applied as stability indicating method for the analysis of carbachol in presence of its metabolite choline, in ophthalmic preparations, in plasma and urine showing good recovery percentage values. MWCNTs based sensor showed some advantages over graphene sensor regarding lower limit of detection (LOD), longer life time and higher selectivity towards carbachol. Statistical comparison of the proposed sensors with the official method showed no significant difference for accuracy and precision.

Direct detection of molecular biorecognition by dipole sensing mechanism.

This work investigates the feasibility of transducing molecular-recognition events into a measurable potentiometric signal. It is shown for the first time that biorecognition of acetylcholine (ACh) can be translated to conformational changes in the enzyme, acetylcholine-esterase (AChE), which in turn induces a measurable change in surface potential. Our results show that a highly sensitive detector for ACh can be obtained by the dilute assembly of AChE on a floating gate derived field effect transistor (FG-FET). A wide concentration range response is observed for ACh (10(-2)-10(-9)M) and for the inhibitor carbamylcholine CCh (10(-6)-10(-11)M). These enhanced sensitivities are modeled theoretically and explained by the combined response of the device to local pH changes and molecular dipole variations due to the enzyme-substrate recognition event.

Case report: acute unintentional carbachol intoxication.

INTRODUCTION: Intoxications with carbachol, a muscarinic cholinergic receptor agonist are rare. We report an interesting case investigating a (near) fatal poisoning. METHODS: The son of an 84-year-old male discovered a newspaper report stating clinical success with plant extracts in Alzheimer's disease. The mode of action was said to be comparable to that of the synthetic compound 'carbamylcholin'; that is, carbachol. He bought 25 g of carbachol as pure substance in a pharmacy, and the father was administered 400 to 500 mg. Carbachol concentrations in serum and urine on day 1 and 2 of hospital admission were analysed by HPLC-mass spectrometry. RESULTS: Minutes after oral administration, the patient developed nausea, sweating and hypotension, and finally collapsed. Bradycardia, cholinergic symptoms and asystole occurred. Initial cardiopulmonary resuscitation and immediate treatment with adrenaline (epinephrine), atropine and furosemide was successful. On hospital admission, blood pressure of the intubated, bradyarrhythmic patient was 100/65 mmHg. Further signs were hyperhidrosis, hypersalivation, bronchorrhoea, and severe miosis; the electrocardiographic finding was atrio-ventricular dissociation. High doses of atropine (up to 50 mg per 24 hours), adrenaline and dopamine were necessary. The patient was extubated 1 week later. However, increased dyspnoea and bronchospasm necessitated reintubation. Respiratory insufficiency was further worsened by Proteus mirabilis infection and severe bronchoconstriction. One week later, the patient was again extubated and 3 days later was transferred to a peripheral ward. On the next day he died, probably as a result of heart failure. Serum samples from the first and second days contained 3.6 and 1.9 mg/l carbachol, respectively. The corresponding urine concentrations amounted to 374 and 554 mg/l. CONCLUSION: This case started with a media report in a popular newspaper, initiated by published, peer-reviewed research on herbals, and involved human failure in a case history, medical examination and clinical treatment. For the first time, an analytical method for the determination of carbachol in plasma and urine has been developed. The analysed carbachol concentration exceeded the supposed serum level resulting from a therapeutic dose by a factor of 130 to 260. Especially in old patients, intensivists should consider intoxications (with cholinergics) as a cause of acute cardiovascular failure.

Effect of early stage of experimental diabetes on vascular functions in isolated perfused kidneys.

The present study investigates the renal vascular responsiveness to vasoactive agents in diabetic rats which present an early stage of renal failure. Adult male Wistar rats were administered alloxan (150 mg kg(-1), s.c.). Seven days later the right kidneys were isolated and perfused. Renal perfusion pressure was measured continuously. Concentration-response curves were plotted for noradrenaline (NA), sodium nitroprusside (SNP) and carbachol. In basal conditions, kidneys from diabetic rats presented a decreased vascular resistance compared with those from control rats. The vasoconstrictor response to NA showed decreased EC50 values in preparations from diabetic rats compared with control ones (EC50 nmols, control: 2.03 +/- 0.44, n = 8; diabetic: 0.84+/-0.18, n = 6, P < 0.05). This enhanced sensitivity to NA could be in line with the decreased glomerular filtration rate and cortical renal plasma flow previously described in vivo in our laboratory (Garcia, Girardi, Ochoa, Torres & Elias, 1998). Vasoconstrictor responses to phenylephrine were not however, different between diabetic and control rat kidneys. This suggests that the increased sensitivity to NA was due to impaired neuronal uptake since phenylephrine is not a substrate for neuronal uptake. After precontraction with phenylephrine, both endothelium-dependent (carbachol) and endothelium independent (SNP) vasodilator agents caused similar response in the preparations taken from the two groups of animals. So, the enhanced sensitivity to NA is not associated with a deficient dilator responsiveness of the renal vasculature. The vasodilator response to carbachol was the same in absence or presence of L-arginine in the perfusate, suggesting no alteration in its availability at this stage of diabetes. Diabetic animals showed increased plasma level of fructosamine and glycosylated haemoglobin (Hb A1c), indicating the presence of early glycated products at this stage of diabetes, which could be involved in a possible structural alteration of the vessels.

1H NMR as an analytical tool for the investigation of hydrolysis rates: a method for the rapid evaluation of the shelf-life of aqueous solutions of drugs with ester groups.

The rate of hydrolysis of esters of primary and secondary alcohols can be determined quickly and easily by 1H NMR in aqueous solution, provided that the water signal is suppressed by the WATR (Water Attenuation by T2 Relaxation) method. To evaluate this approach, Arrhenius plots have been constructed for hydrolysis of acetylcholine, carbachol and atropine, and the effect of pH on the hydrolysis of procaine has been determined over a limited range. The results agree well with literature values for rate constants.

Restricted diffusion and stability of carbachol-fluorescent nanospheres in-vivo.

Mapping neuronal populations that induce behavioral state changes after pharmacological activation requires discrete localization of drug injection sites, and is limited by widespread diffusion of molecular drugs. Nanospheres with diameters of 50-100 nm can reduce diffusion significantly because of their relatively large sizes. The cholinergic agonist carbachol was radiolabeled with methyl14C and incorporated within a latex nanosphere delivery system (LNDS). We quantitatively compared diffusion of 14C-carbachol within these nanospheres with that of free 14C-carbachol, demonstrating approximately ten-fold reduced radial diffusion by nanospheres 10 min to 24 h post-injection; approximately 90% of injected radioactivity was restricted to regions within approximately 100-150 microns and 1400-1500 microns respectively. Thus, incorporation of active agents such as drugs within nanospheres dramatically increases the precision of their delivery in-vivo (here about 1,000-fold by volume).

Persistence of enhanced aerosol deposition in the lung after recovery from carbachol-induced airway obstruction.

Time course recovery from induced airway obstruction by carbachol infusion (CI; 0.2 microgram.kg-1.min-1 for 40 min), carbachol aerosol (CA; 10 breaths of 2% solution), and histamine aerosol (HA; 25-50 breaths of 5% solution) challenge was investigated in conscious sheep (n = 6 each). Total lung aerosol deposition and airway caliber as assessed by pulmonary airflow resistance (RL) were measured every 20-30 min up to 4 h after the challenges. Aerosol deposition was measured by monitoring aerosol concentration continuously with a laser aerosol photometer while the sheep rebreathed 1.0-micron-diam inert oil droplets delivered by a 0.25-liter bag-in-box system driven by a respiratory pump at a breathing frequency of 30 breaths/min. Total accumulated deposition at the fifth breath (AD5) as percentage of the initial aerosol concentration was determined and used as an aerosol deposition index. Percent changes in AD5 from baseline were compared with corresponding changes in RL. Both RL and AD5 increased after Cl, CA, and HA: 192-477% for RL and 23-44% for AD5 (P less than 0.05). Mean RL return to baseline values 1 h after CI and HA and 2 h after CA. Mean AD5 returned to baseline at 1 h post-HA. In contrast, mean AD5 remained elevated for 2-4 h after CI and CA (P less than 0.05), and the increased AD5 could not be reversed by a bronchodilator aerosol. The persistence of enhanced aerosol deposition long after the return of RL to baseline suggests that complete recovery of airway conditions after CI and CA takes much longer than predicted by RL.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosol deposition in the lung with asymmetric airways obstruction: in vivo observation.

Both the total and regional aerosol deposition were measured in six adult sheep before and after an induction of asymmetric airway obstructions, either by local instillation of carbachol solution (CS, 0.1%) distal to the right main bronchus or inhalation challenge of the right lung with carbachol aerosol (CA, 10 breaths). Total lung deposition was determined by monitoring inert monodisperse aerosols [1.0 micron mass median aerodynamic diam (MMAD)] breath-by-breath, at the mouth, by means of a laser aerosol photometer. Cumulative aerosol deposition over the first five breaths as a percent of the initial aerosol concentration (AD5) was used as a deposition index. Regional deposition pattern was determined by scintigraphic images of sulfur-colloid aerosol (1.5 microns MMAD) tagged with 99mTc. Radioactivity counts in the right (R) and left lung (L) were expressed as a percent of the whole lung count. Half-lung AD5 was then determined by multiplying AD5 by fractional radioaerosol depositions in R or L. Pulmonary airflow resistance (RL mean +/- SE), as determined by an esophageal balloon technique, increased by 111 +/- 28 and 250 +/- 96% after CA and CS, respectively (P less than 0.05). AD5 also increased in all the sheep tested by 29 +/- 3 and 52 +/- 8%, respectively, after CA and CS (P less than 0.05). Radioaerosol deposition pattern was even at base line (R/L = 51:49) but shifted toward the unchallenged L after CS (R/L = 40:60). Deposition pattern after CA was variable: a shift toward L in three, no change in one, and a shift toward the R lung in two sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic brainstem mechanisms of REM sleep in the rat.

Injection of carbachol into the brainstem of rats produced an increase in rapid eye movement (REM) sleep which was site- and dose-dependent. Effective locations for carbachol to stimulate REM sleep included the pontine reticular formation at the level of the trigeminal motor nucleus and the dorsal parabrachial area in the caudal midbrain. The carbachol effect in the caudal pons was dose-dependent. Additionally, this effect was blocked by concomitant administration of the muscarinic antagonist atropine. Control experiments suggested that the drug-induced phenomenon appeared to be an increase in normal physiological REM sleep.

IR spectrophotometric assay of carbachol solutions.

Carbachol ophthalmic solutions can be assayed by evaporating a measured volume, dissolving the residue in methanol, and scanning the carbonyl stretching frequency in an IR spectrophotometer using a cell with calcium fluoride windows. Methylcellulose and other formulation vehicle components do not interfere. The method is stability indicating with respect to hydrolysis. It affords a recovery of 99.5+/-0.51% (RSD).