Serum Adipokines, Growth Factors, and Cytokines Are Independently Associated with Stunting in Bangladeshi Children.

Growth in young children is controlled through the release of several hormonal signals, which are affected by diet, infection, and other exposures. Stunting is clearly a growth disorder, yet limited evidence exists documenting the association of different growth biomarkers with child stunting. This study explored the association between different growth biomarkers and stunting in Bangladeshi children. A quasi-experimental study was conducted among 50 stunted (length-for-age Z-score (LAZ) < -2 SD) and 50 control (LAZ ≥ -2 SD) children, aged 12-18 months, residing in a Bangladeshi slum. The enrolled stunted children received an intervention package, which included food supplementation for three months, psychosocial stimulation for six months, and routine clinical care on community nutrition center at the study field site. The controls received routine clinical care only. All children were clinically screened over the study period. Length, weight, fasting blood and fecal biomarkers were measured. All biomarkers levels were similar in both groups except for oxyntomodulin at enrolment. Leptin (adjusted odds ratio, AOR: 4.0, p < 0.01), leptin-adiponectin ratio (AOR 5.07 × 108, p < 0.01), insulin-like growth factor-1 (IGF-1) (AOR 1.02, p < 0.05), and gamma interferon (IFN-γ) (AOR 0.92, p < 0.05) levels were independently associated with stunting at enrolment. Serum leptin, leptin-adiponectin ratio, interleukin-6 (IL-6), IL-10, tumor necrosis factor-alpha (TNF-α), and fecal alpha-1-antitrypsin (AAT) levels increased significantly (p < 0.001), while IFN-γ levels significantly decreased among stunted children after six months of intervention. Leptin, leptin-adiponectin ratio, IGF-1, and IFN-γ are independently associated with stunting in Bangladeshi children. This trial was registered at clinicaltrials.gov as NCT02839148.

Determination of flumazenil in serum by liquid chromatography-mass spectrometry: Application to kinetics study in acute diazepam overdose.

BACKGOUND/AIM: Flumazenil is benzodiazepine receptor antagonist. It has been studied for a various indications, including reversal of sedation after surgery or diagnostic procedures, awakening of comatose patients in benzodiazepine overdose, or for symptomatic treatment of hepatic encephalopathy. Some drugs, like theophylline, may prolong its elimination half-life. Considering the long half-life of diazepam and its metabolites, concomitant use of theophylline may reduce the need for repeated dosing of flumazenil in patients with acute diazepam poisoning. The aim of this study was to introduce a reliable and accurate method for determining the concentration of flumazenil after therapeutic application in patients with acute poisoning, and using that method to assess whether the kinetics of flumazenil change in the presence of aminophylline (combination of theophylline and ethylenediamine in a 2:1 ratio) applied as concomitant therapy. METHODS: Blood samples from patients with acute diazepam poisoning that received flumazenil at the dose of 0.5 mg, or the same dose with 3 mg/kg of body weight of aminophylline, were collected 1, 3, 10, 30, 60, 120 and 240 min after its intravenous administration. Samples were prepared by solid-phase extraction on Oasis HLB cartridges with ethylacetate as extracting agens. Flumazenil was determined by liquid chromatography with mass spectrometry (LC-MS) in single ionmonitoring mode at m/z 304. Separation of flumazenil from matrix compound was performed on Lichrospher RP-8 column usingthe mixture of acidic acetonitrile and 20 mM of ammonium acetatein water (55 : 45) as a mobile phase. RESULTS: The applied analitycal method showed excellent recovery (94.65%). The obtained extracts were much cleaner than the extracts obtained by the sameextractant in the process of liquid-liquid extraction. The limit ofdetection of the LC-MS method described in this paper was 0.5 ng/mL and the limit of quantitation was 1 ng/mL. In the patientstreated with both flumazenil and aminophylline, the eliminationconstant for flumazenil was significantly lower and the elimination half-life was longer (p < 0.05) in comparison with the same parameters in.the patients who received flumazenil alone. CONCLUSION: The applied LC-MS method for the determination of flumazenil in serum samples of patients with acute diazepam poisoning is rapid, sensitive, precise and specific. Concomitant use with theophylline significantly prolonged elimination of flumazenil during the treatment of acute poisonings with diazepam.

MRI-based correction for partial-volume effect improves detectability of intractable epileptogenic foci on 123I-iomazenil brain SPECT images.

UNLABELLED: (123)I-Iomazenil brain SPECT has been used for the detection of epileptogenic foci, especially when surgical intervention is considered. Although epileptogenic foci exhibit a decrease in (123)I-iomazenil accumulation, normal cerebral cortices often exhibit similar findings because of thin cortical ribbons, gray matter atrophy, or pathologic brain structures. In the present study, we created (123)I-iomazenil SPECT images corrected for gray matter volume using MRI and tested whether the detectability of the epileptogenic foci improved. METHODS: Seven patients (1 male patient and 6 female patients; mean age +/- SD, 34 +/- 17 y) with intractable epilepsy were surgically treated by resecting the cerebral cortex after surface electroencephalography. Histopathologic examination of the resected specimens and a good outcome after surgery indicated that the resected lesions were epileptogenic foci. These patients underwent (123)I-iomazenil SPECT and 3-dimensional T1-weighted MRI examinations before their operations. Each SPECT image was coregistered to the corresponding MR image, and its partial-volume effect (PVE) was corrected on a voxel-by-voxel basis with a smoothed gray matter distribution image. Four nuclear medicine physicians visually evaluated the (123)I-iomazenil SPECT images with and without the PVE correction. The SPECT count ratio of the suspected focus to the contralateral cerebral cortex was evaluated as an asymmetry index (%) based on the volume of interest. RESULTS: The sensitivity, specificity, and accuracy of focus detection by visual assessment were higher after PVE correction (88%, 99%, and 98%, respectively) than before correction (50%, 92%, and 87%, respectively). The mean asymmetry index for the surgically resected lesions was significantly higher on the PVE-corrected SPECT images (22%) than on the PVE-uncorrected ones (16%) (P = 0.006). CONCLUSION: MRI-based PVE correction for (123)I-iomazenil brain SPECT improves the sensitivity and specificity of the detection of cortical epileptogenic foci in patients with intractable epilepsy.

Comparison of plasma input and reference tissue models for analysing [(11)C]flumazenil studies.

A single-tissue compartment model with plasma input is the established method for analysing [(11)C]flumazenil ([(11)C]FMZ) studies. However, arterial cannulation and measurement of metabolites are time-consuming. Therefore, a reference tissue approach is appealing, but this approach has not been fully validated for [(11)C]FMZ. Dynamic [(11)C]FMZ positron emission tomography scans with arterial blood sampling were performed in nine drug-free depressive patients and eight healthy subjects. Regions of interest were defined on co-registered magnetic resonance imaging scans and projected onto dynamic [(11)C]FMZ images. Using a Hill-type metabolite function, single (1T) and reversible two-tissue (2T) compartmental models were compared. Simplified reference tissue model (SRTM) and full reference tissue model (FRTM) were investigated using both pons and (centrum semiovale) white matter as reference tissue. The 2T model provided the best fit in 59% of cases. Two-tissue V(T) values were on average 1.6% higher than 1T V(T) values. Owing to the higher rejection rate of 2T fits (7.3%), the 1T model was selected as plasma input method of choice. SRTM was superior to FRTM, irrespective whether pons or white matter was used as reference tissue. BP(ND) values obtained with SRTM correlated strongly with 1T V(T) (r=0.998 and 0.995 for pons and white matter, respectively). Use of white matter as reference tissue resulted in 5.5% rejected fits, primarily in areas with intermediate receptor density. No fits were rejected using pons as reference tissue. Pons produced 23% higher BP(ND) values than white matter. In conclusion, for most clinical studies, SRTM with pons as reference tissue can be used for quantifying [(11)C]FMZ binding.

Population pharmacokinetic analysis for simultaneous determination of B (max) and K (D) in vivo by positron emission tomography.

PURPOSE: Changes in GABA(A)-receptor density and affinity play an important role in many forms of epilepsy. A novel approach, using positron emission tomography (PET) and [C-11]flumazenil ([C-11]FMZ), was developed for simultaneous estimation of GABA(A)-receptor properties, characterized by B (max) and K (D). PROCEDURES: Following an injection of [C-11]FMZ (dose range: 1-2,000 mug) to 21 rats, concentration time curves of FMZ in brain (using PET) and blood (using HPLC-UV) were analyzed simultaneously using a population pharmacokinetic (PK) model, containing expressions to describe the time course of the plasma concentration (including distribution to the body), the brain distribution, and the specific binding within the brain. RESULTS: Application of this method in control rats resulted in estimates of B (max) and K (D) (14.5 +/- 3.7 ng/ml and 4.68 +/- 1.5 ng/ml, respectively). CONCLUSIONS: The proposed population PK model allowed for simultaneous estimation of B (max) and K (D) for a group of animals using single injection PET experiments per animal.

Analysis of microsomal metabolic stability using high-flow-rate extraction coupled to capillary liquid chromatography-mass spectrometry.

A method is described for on-line high-speed extraction of microsomal samples and analysis by capillary liquid chromatography-mass spectrometry (LC-MS) for the determination of metabolic stability in connection with the development of positron emission tomography (PET) tracers. The method allowed direct injections of large sample volumes at a fast extraction rate, providing a gain in both sensitivity and sample preparation time. The calibration curve of the test compound flumazenil (Ro 15-1788) was linear in the concentration range of 1-150 nM, with a correlation coefficient exceeding 0.999. The accuracy of the method ranged from 98 to 101%. A high precision was obtained, with mean intra-assay and inter-assay relative standard deviations of at most 1.4 and 1.5%, respectively, for quality control (QC) samples. The extraction efficiency was determined to be 99.4%, the total recovery 96% and the carryover to

Assessment of [18F]fluoroethylflumazenil metabolites using high-performance liquid chromatography and tandem mass spectrometry.

A simple procedure using HPLC and tandem mass spectrometry has been developed for the determination of fluoroethylflumazenil metabolites. Samples were precipitated with acetonitrile, evaporated to dryness followed by reconstitution with methanol. As mobile phase, 50 mM ammonium formate-methanol (58:42, v/v) was used. The method is valid both for cold and radiolabelled metabolites. Various cold metabolites (hydroxylated and/or dealkylated) were identified in rat and human microsome preparations. Radiolabelled metabolites arise from two or more transformations including hydroxylation. The methodology developed can be applied for further characterisation of metabolites, and for the determination of non metabolised [18F]fluoroethylflumazenil in routine clinical analysis.

A fatality due to moclobemide.

A fatality due to ingestion of the antidepressant drug moclobemide is reported. Moclobemide is a selective and reversible inhibitor of monoamine oxidase type A. Previous reports have suggested that it is a safe drug even when taken in large quantities. The few reported fatalities have all been ascribed to serotonin syndrome, due to an interaction between moclobemide and other serotonergic agents. A 48-year-old woman with a history of depression and suicide attempts was found deceased at home. Autopsy revealed no evidence of significant natural disease or injury. Toxicologic analysis was performed and drug levels measured by capillary gas chromatography. The blood concentration of moclobemide was 137 mg/L and the liver concentration was 432 mg/kg. Low levels of diazepam, nordiazepam, and trifluoperazine were also detected. Death was considered to be due to acute poisoning by moclobemide. This case report is the first, to our knowledge, where death has been attributed to the toxic effects of moclobemide alone.

Evaluation of ultrafiltration for the free-fraction determination of single photon emission computed tomography (SPECT) radiotracers: beta-CIT, IBF, and iomazenil.

An ultrafiltration system was evaluated for the free-fraction measurement of SPECT radiotracers (beta-CIT, IBF, and iomazenil) used in functional brain imaging. The effect of temperature, storage, centrifugal force, tracer concentration, and percentage filtered demonstrated a relative error of < 9%. As a result of the minimal temperature effect, 25 degrees C was employed for all measurements. A comparison of the ultrafiltration system with equilibrium dialysis revealed < 5% difference for beta-CIT and iomazenil, but 16% for IBF. Additionally, the time and ease of operation considerably favored the ultrafiltration system. The precision quantitated by repetition was < 6% for between-run and within-run variability. In conclusion, ultrafiltration provided rapid results, demonstrated minor analytical errors, revealed generally good correlation with equilibrium dialysis, and allowed excellent precision.

Midazolam and flumazenil pharmacokinetics and pharmacodynamics following simultaneous administration to human volunteers.

Resedation after antagonism of midazolam sedation with flumazenil may occur because some individuals have rapid elimination of flumazenil but slow elimination of midazolam. To determine whether there are parallel or divergent rates of elimination of the two drugs between individuals, the pharmacokinetic profiles of midazolam and flumazenil were studied simultaneously in 12 adult male volunteers. Free drug concentration data for the two drugs were incorporated into a receptor occupancy model and psychomotor testing was performed and correlated with receptor occupancy. Variation was found between individuals in the pharmacokinetics of the two drugs. There were significant correlations between Cltot (P < 0.01) but not in t1/2 alpha, t1/2 beta, Vc, or VDss. In individuals, midazolam elimination half-life ranged from less than half that of flumazenil to more than three times that of flumazenil. There was a relatively poor, although statistically significant linear correlation found between calculated receptor occupancy and critical flicker fusion frequency, r = 0.50, P < 0.01, and linear analogue scales of sedation r = 0.56, P < 0.005; and anxiolysis, r = 0.54, P < 0.005. There is divergence in the disposition and elimination of midazolam and flumazenil in some individuals. A benzodiazepine receptor occupancy model is useful for predicting the consequent differences in clinical effect when the drugs are given together.

Potential use of a radioreceptor assay for the determination of benzodiazepine compounds in serum.

The clinical monitoring and intensive care of patients who have taken an overdose of a benzodiazepine require rapid and quantitative methods to assess benzodiazepine concentrations in biological fluids. A radioreceptor assay (RRA) permits the simultaneous measurement of the benzodiazepine molecules that bind to the receptor, providing a total estimate of all pharmacologically active forms of the drug(s) (parent drug and active metabolites). This study describes the development of an RRA for the determination of benzodiazepine compounds in serum using a lyophilized bovine brain cerebral cortex receptor preparation. The standard curves and the sensitivity of this RRA are determined for 20 different benzodiazepines and the specific antagonist flumazenil. The sensitivity ranges from 5 ng/mL for clonazepam to 3500 ng/mL for chlordiazepoxide. The IC50 values are significantly correlated (r = 0.81) with the lowest recommended therapeutic concentrations. The advantages and disadvantages of this RRA are discussed and compared with those of other chromatographic and immunological methods.

Simultaneous determination of flumazenil, midazolam and metabolites in human biological fluids by liquid chromatography.

A simple procedure for the simultaneous determination of flumazenil, midazolam, 1-hydroxymethylmidazolam and 4-hydroxymidazolam in plasma or urine in surgical patients is described. The assay involves a preliminary extraction of the drugs, metabolites and internal standard (flurazepam) from biological fluid into an organic solvent mixture (dichloromethane-diethyl ether, 40:60 v/v). The extract was evaporated to dryness at 40 degrees C under a gentle stream of nitrogen. The residue was re-dissolved in distilled methanol (80 microliters) and a 30-microliters aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile phase (32% acetonitrile in 0.004 M sodium hydrogenphosphate buffer containing 1 ml of triethylamine and adjusted to pH 7.2) at a flow-rate of 1.5 ml/min on a 30-microns C8 precolumn linked to a 4-microns Nova-pak C18 cartridge column (100 mm x 8 mm I.D.) at ambient temperature (25 degrees C). The eluate was detected at 220 nm.

Reversal of midazolam sedation with flumazenil.

OBJECTIVE: To demonstrate the efficacy of flumazenil in reversing the sedative action of midazolam in ventilated intensive care patients. DESIGN: Prospective, double-blind randomized study. SETTING: ICU of a tertiary, university-affiliated teaching hospital. PATIENTS: Thirty ICU patients requiring artificial ventilation for greater than 12 hrs were studied. INTERVENTIONS: All patients received a midazolam infusion for sedation. Twenty-nine patients received supplementary narcotics. At the end of the sedation period, either flumazenil or placebo was administered to all the patients in a double-blind, randomized fashion, and the effects were observed. MEASUREMENTS AND MAIN RESULTS: Sedation levels were measured hourly during the infusion; at the end of the infusion; and at 5, 15, 30, 60, and 120 mins after cessation of the midazolam infusion. Midazolam concentrations in serum were measured at the time of cessation of the midazolam infusion and at 30, 60, and 120 mins later. Reversal of sedation was observed in 14 of 15 patients who received flumazenil, and resedation occurred in seven of these patients. Reversal was not seen in any of the patients who received placebo. Midazolam serum concentrations were similar in both groups. CONCLUSION: Flumazenil in a dose of 0.15 mg is a safe drug that reverses the sedative effect of midazolam.

Plasma determination of flumazenil, a benzodiazepine antagonist, by immunotoxicology and by capillary gas chromatography/mass spectrometry.

The method presented describes a sensitive and specific quantitative assay for the determination of flumazenil in human plasma. Flumazenil and an internal standard, midazolam, are isolated by a basic extraction. The final extract is separated on a 25-m BP-1 capillary column and drugs are detected by selected ion monitoring at m/z 229 and m/z 310 for flumazenil and the internal standard, respectively. The minimum detectable quantity is 1.0 ng/mL, for flumazenil in plasma. Coefficients of variation for within-run data were less than 6%.

HPLC quantification of zolpidem and prothipendyl in a voluntary intoxication.

Zolpidem, a recently developed sleep inducer, and prothipendyl, a neuroleptic azaphenothiazine, were involved in a voluntary intoxication along with ethanol. After administration of flumazenil, a specific benzodiazepines antagonist, respiratory depression was corrected. HPLC with UV detection methods after selective extraction were developed to measure simultaneously prothipendyl and zolpidem without flumazenil interaction. These methods could be applied in drug monitoring and in emergency toxicology.

The stability of flumazenil in infusion solution.

Flumazenil, the first benzodiazepine antagonist to be marketed, is available only for intravenous injection (0.1 mg/ml). The half-life of the compound in plasma is short compared with that of the conventional benzodiazepines, and administration of the drug as a continuous infusion is therefore frequently required. However, the stability of flumazenil in infusion solution has not previously been known, which has meant that controlled studies of the drug in this administration form have been difficult to perform. In the present study, infusion solutions of flumazenil in concentrations of 1.0 and 5.0 micrograms/ml were produced and stored for periods of up to 9 months. The concentrations of the drug in the different solutions were determined by gas chromatography at defined intervals, and were found not to change during the study period. We conclude that the stability of flumazenil in infusion solution is satisfactory.

Rapid determination of the benzodiazepine antagonist flumazenil, Ro 15-1788, by high performance liquid chromatography.

A sensitive (2 ng/mL) and specific method for the determination of the benzodiazepine antagonist Ro 15-1788 or flumazenil is described. Following a simple extraction, the compound is analyzed by reversed phase high performance liquid chromatography (HPLC) and detection at 245 nm. The method was applied to plasma specimens collected from patients receiving a single dose of this drug.