Development of New Health Risk Assessment of Nanoparticles: EPA Health Risk Assessment Revised.

The concentration of nanoparticles in the ambient air can lead to induced toxicities; however, it appears that nanoparticles' unique properties are completely omitted when assessing health risks. This paper aims to enhance the EPA health risk assessment by incorporating two new variables that consider the size of nanoparticles: the toxicity multiplier and the size multiplier. The former considers the qualitative aspect of the size of particles within a concentration, whilst the latter takes into account the effects associated with the number of particles of the specific i-th size distribution interval. To observe the impact of the new variables, a case study was performed. The studied element was cadmium, which was measured using ICP-MS to discover concentrations of size fractions, ranging from <15.1 to <9830 nm. Next, the cadmium concentration is assessed using both the current state-of-the-art method and the proposed method with adjustments. Based on the new approach, the final risk was 1.1 × 10−5, which was almost 24 times higher compared with the current method. The contribution of nanoparticles to the risk value grew from barely 6% to an alarming 88%. Therefore, the enhanced method can lead to more realistic results when assessing the health risks of nanoparticles.

Simultaneous HPLC determination of tolbutamide, phenacetin and their metabolites as markers of cytochromes 1A2 and 2C6/11 in rat liver perfusate.

A new, simple, rapid, sensitive, and repeatable reversed-phase HPLC method was developed and validated for the simultaneous determination of tolbutamide, phenacetin and their metabolites in rat liver perfusate. Chlorpropamide was used as an internal standard to ensure the precision and accuracy of this method. Analytes were extracted into diethyl ether using a two-step liquid-liquid extraction. A C18 analytical column and a mobile phase composed of acetonitrile and potassium phosphate buffer were used for the chromatographic separation with UV detection. Limits of detection varied between 20 and 46ng/mL for phenacetin, tolbutamide and their metabolites. The overall extraction recovery for the analytes varied from 65.4% in paracetamol to 88.0% in tolbutamide for concentrations within the expected range of concentrations from previous experimental samples. In terms of precision, the intra- and inter-day variation at three different concentrations in all analytes never exceeded 7.6 and 11.4%, respectively. This method is applicable for the modeling and description of possible pharmacological interactions on rat cytochromes P450 1A2 and 2C6/11 or can be used for in vitro evaluation of both cytochromes 1A2 and 2C.

Superiority of thoracoscopic sympathectomy over medical management for the palmoplantar subset of severe hyperhidrosis.

Severe hyperhidrosis is a disabling disorder whose management is controversial. Medical treatment consists of topical aluminum chloride, oral anticholinergics, ionotophoresis, and botulinum toxin A (Botox) injections. Despite the minimally invasive nature of thoracoscopic sympathectomy, there is a common perception that surgery is only a "last resort." The palmoplantar subtype of hyperhidrosis is particularly problematic for patients professionally and socially. The purpose of our study was to determine the safety, efficacy, and side effects of the various medical treatments vs. bilateral thoracoscopic sympathectomy (BTS) for palmoplantar hyperhidrosis. Consecutive patients (n = 192) were selected based on massive palmar sweating, similar level of plantar sweating, bimodal onset in early childhood or puberty, and exacerbation with ordinary hand lotion. A prospective cohort of 47 patients underwent medical treatment with their responses monitored on a prospective basis, and 145 patients underwent retrospective evaluation of their medical treatment based on their histories. Patients whose medical treatments failed or resulted in intolerable side effects were offered outpatient BTS surgery at the T2-T3 level. Of the 47 prospective patients, 46 received topical aluminum chloride, 40 anticholinergics, six iontophoresis, and 45 BTS surgery. Only one patient was successfully treated with aluminum chloride (2.2%) and one successfully treated with anticholinergics (2.5%), and these did not undergo surgery. Iontophoresis was not successful in any prospectively followed patient. BTS was effective in curing palmar hyperhidrosis in 100% of patients. The superiority of BTS vs. topical aluminum chloride, anticholinergics, and iontophoresis to successfully treat palmar hyperhidrosis was highly statistically significant (p < 0.001). For the retrospective group of 145 patients, 89 had been treated with topical aluminum chloride, 38 with oral anticholinergics, 31 with iontophoresis, eight with Botox, one with no medical treatment, and 144 with BTS surgery. All medical treatments failed with the exception that one patient was satisfied with anticholinergic treatment (2.6%), and this patient did not undergo BTS. BTS was successful in curing bilateral palmar hyperhidrosis in 99.3% (one unilateral failure due to adhesions). BTS was superior in treating palmar hyperhidrosis compared to aluminum chloride, anticholinergics, iontophoresis, and Botox (p < 0.001). The medically treated patients suffered significant side effects ranging from local stinging, cracking, and blistering to xerostomia, xerophthalmia, and blunted mentation. Overall, compensatory hyperhidrosis (CH) was present in 56% of patients undergoing BTS, but only 3.2% of BTS patients had severe CH with significant discomfort; all were men. There were no other significant operative complications. The safety and overwhelming efficacy of BTS compared to medical management of severe palmoplantar hyperhidrosis is demonstrated. Rather than being a "last resort," BTS can be confidently recommended as first-line treatment for the typical, severe form of palmoplantar hyperhidrosis.

Development of electrophoretically mediated microanalysis method for the kinetics study of flavin-containing monooxygenase in a partially filled capillary.

An electrophoretically mediated microanalysis method with a partial filling technique was developed for flavin-containing monooxygenase, form 3 (FMO3). The in-line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 7.4) whereas 150 mM phosphate buffer (pH 3.3) was used as a background electrolyte. A long plug of cofactor NADPH dissolved in reaction buffer was hydrodynamically injected into a fused-silica capillary, followed by enzyme and substrate solution. The reaction was initiated at 37 degrees C in the thermostated part of the cartridge by the application of 9 kV for 0.9 min. The voltage was turned off to increase the product amount (zero-potential amplification) and again turned on at a constant voltage of 10 kV to elute all the components. Direct detection was performed at 191 nm. The developed electrophoretically mediated microanalysis method was applied for the kinetics study of FMO3 using clozapine as a substrate probe. A Michaelis-Menten constant (K(m)) of 410.3 microM was estimated from the corrected peak area of the product, clozapine N-oxide. The calculated value of the maximum reaction velocity (V(max)) was found to be 1.86 nmol/nmol enzyme/min. The acquired FMO3 kinetic parameters are in accordance with the published literature data.

Application of micellar electrokinetic capillary chromatography for evaluation of inhibitory effects on cytochrome P450 reaction.

The major aim of this work is to demonstrate the applicability of micellar electrokinetic capillary chromatography with SDS based pseudostationary phase for the screening of cytochrome P450 inhibitors. In contrast with the other capillary electrophoresis modes the cytochrome P450 reaction mixture thus could be used for the analysis without any pre-treatment. Cytochrome P450 2C9, one of the most important isoforms in human liver, was chosen as a model example for this study in combination with diclofenac as a probe substrate. The inhibitory effect on the given cytochrome P450 reaction was evaluated for two inhibitors with different inhibition potential - strong inhibitor sulfaphenazole and moderate inhibitor ketoconazole. As a result 50% inhibitory concentrations IC(50) and inhibition constants K(i) were evaluated; their values for both inhibitors were in a good agreement with the literature data determined by different methods.

HPLC determination of midazolam and its three hydroxy metabolites in perfusion medium and plasma from rats.

A new, simple, rapid, sensitive, and repeatable isocratic reverse-phase HPLC method was developed and validated for simultaneous determination of midazolam and its main three hydroxylated metabolites, i.e. 1'-hydroxymidazolam, 4-hydroxymidazolam, and 1',4-dihydroxymidazolam in rat liver perfusate and also plasma. Diazepam was used as an internal standard to ensure precision and accuracy of this method. Analytes were extracted from alkalinized samples into diethyl ether using single-step liquid-liquid extraction. A C18 analytical column and a mobile phase composed of acetonitrile and sodium acetate buffer were used for the chromatographic separation with UV detection. Limits of detection varied between 7.9 and 19.6 microg/L for midazolam and its hydroxy metabolites. The overall recovery for the analytes exceeded 92%, for concentrations twice the limits of detection. The intra- and inter-day precision at three different concentrations never exceeded 8 and 11% variation, respectively. This method is applicable for modeling and description of possible pharmacological interactions on rat (CYP3A1/2) or human (CYP3A4/5) cytochrome P450 enzymes.

Study of recombinant cytochrome P450 2C9 activity with diclofenac by MEKC.

Cytochrome P450 2C9 (CYP2C9) is one of the most important isoforms in human liver involved in the metabolism of a large number of therapeutic agents. The aim of this paper is to demonstrate the applicability of CE for the determination of the enzymatic activity of CYP2C9 with diclofenac as a probe substrate. MEKC with SDS as a pseudostationary phase was used for this purpose. Compared to other assays, the MEKC-based method is rapid, can be automated and requires only a small quantity of enzymes and substrate. Moreover, the enzymatic reaction can be monitored with high sensitivity and repeatability even when the reaction mixture is used for the analysis without any pretreatment. The kinetic study on the given enzymatic reaction was also performed since the basic characterization of drug biotransformation generally begins with the enzyme kinetic analysis of metabolite formation. As a result, the Michaelis constant and maximum reaction velocity were evaluated, the values 3.44 +/- 0.45 microM and 19.78 +/- 0.76 nmol min(-1) nmol(-1), respectively, were in agreement with the literature data. On the other hand, a slight deviation from typical Michaelis-Menten kinetics with a weak positive cooperativity was found at diclofenac concentrations below 2 microM. The same atypical kinetic behavior of CYP2C9 was also observed by other authors.