To create a cleanroom controlled environment using a mobile air decontamination unit for the preparation of antineoplastic drugs.

OBJECTIVES: To use a mobile air decontamination unit (MADU) for a microbial destruction and decreased particle burden making a cleanroom controlled environment in a Centralized Chemotherapy Preparation Unit (CCPU). Good manufacturing practices (GMP) in France specify that the ambient air in the vicinity of a class III biosafety cabinet (isolator) complies with air cleanliness ISO 8 level in CCPU. This guideline has a significant impact because implementing a dedicated air handling unit (AHU) brings some engineering constraints and generates substantial additional costs. METHODS: Authors have previously studied some technical and economical aspects to evaluate the feasibility of the MADU option. RESULTS: Using a MADU was the chosen option. Qualification of the CCPU showed that results were in compliance with the French GMP. After one year of use, the efficiency of the MADU was confirmed. According to these results, using a MADU constitutes a beneficial option for CCPU previously equipped with an isolator when compared to renovation work involving a standard built-in AHU.

Determination of acid base balance and oil water partition coefficient of an atopy patch test of levofloxacin.

Levofloxacin, a quinolone used in the treatment of an upper respiratory tract infection is involved in skin reactions. Patch tests are helpful for the aetiological diagnosis. Unfortunately, they are known for their lack of sensibility. A weak transcutaneous penetration due to the physicochemical characteristics of tested drugs is a well-known cause of false negative results but these characteristics are usually unknown. The aim of the present study was to evaluate physicochemical parameters by potentiometric method in order to optimize the transcutaneous penetration of a preparation for patch test. This method is applicable to a medium such as the preparation for patch test. In order to value the stability of the preparation, qualitative and quantitative analysis of the level of levofloxacin was effectuated. In our study, spectrophotometric and potentiometric data at T0 and six months later with the preparation showed the stability of the preparation. The proportions of non-ionized (45%) and ionized levofloxacin (55%) were not modified. Further experiments are needed to choose the minimal efficient dose of the suspected molecule able to detect a positive reaction in cases of allergic reaction. In addition, this method could be useful in qualitative and quantitative control of preparations made at hospital.

Comparison of three methods for oxidative stress-induced potassium efflux measurement.

The aim of the study was to compare the potassium efflux measurements (flame photometry (FP), specific electrode (SE) and atomic absorption photometry (AAP)) using a model of erythrocytes exposed to an oxidative stress in various conditions of osmolarity. Human erythrocytes were incubated in 3 different values of osmolarity and in the presence of 50mM AAPH, potassium efflux was measured by FP, SE and AAP at t=0 and every 30min for 2h. These methods were similar for the measurement of global potassium efflux. However, SE detected important amounts of potassium at the beginning of the experiment or in absence of AAPH in comparison with AAP and FP. It is noteworthy that these different methods of measurements were not altered by the osmolarity. FP and AAP make it possible to study the potassium efflux during oxidative stress while SE should be used only for global measurements.

Effects of an oxidative stress on human hemoglobin: a multiwavelength visible spectrometry study.

This study was carried out to investigate hemoglobin behavior and the role of cell membrane during oxidative stress of human red blood cells induced by a water-soluble radical initiator, 2,2'-azobis(amidino-propane) hydrochloride (AAPH) and compare the observed data to the one obtained with purified human haemoglobin solution. The different forms of hemoglobin were identified and quantified by multiwavelength visible spectrometry using multiple linear regression analysis. Hemolysis was quantified by the Drabkin method. Oxidative stress on purified hemoglobin solutions induced an early formation of Hb(+). In intact erythrocytes, no modified form of haemoglobin was found. Only the hemoglobin released by hemolysis in the extracellular medium was notified in the same way as purified haemoglobin. Thus, the cell membrane appears to protect intraerythrocytic hemoglobin toward an extracellular oxidative stress. Oxidative stress-induced by hemolysis does not seem to be due to changes in intraerythrocytic hemoglobin forms.

The influence of extracorporeal circulation on the susceptibility of erythrocytes to oxidative stress.

Extracorporeal circulation (ECC), a necessary and integral part of cardiac surgery, can itself induce deleterious effects in patients. The pathogenesis of diffuse damage of several tissues is multifactorial. It is believed that circulation of blood extracorporeally through plastic tubes causes a whole body inflammatory response and a severe shear stress to blood cells. The aim of this study was to evaluate the level of oxidative stress and its deleterious effect on red blood cell (RBC) before (pre-ECC), immediately after (per-ECC) and 24 h after an ECC (24 h post-ECC). Several indicators of extracellular oxidative status were evaluated. The ascorbyl free radical (AFR) was directly measured in plasma using electron spin resonance (ESR) spectroscopy and expressed with respect to vitamin C levels in order to obtain a direct index of oxidative stress. Allophycocyanin assay was also used to investigate the plasma antioxidant status (PAS). Indirect parameters of antioxidant capacities of plasma such as vitamin E, thiol and uric acid levels were also quantified. RBC alterations were evaluated through potassium efflux and carbonyl levels after action of AAPH, a compound generating carbon centered free radicals. No changes in plasma uric acid and thiols levels were observed after ECC. However, vitamin E levels and PAS were decreased in per-ECC and 24h post-ECC samples. Vitamin C levels were significantly lower in 24 h post-ECC and the AFR/ vitamin C ratio was increased. Differences in results had been noted when measurements took account of hemodilution. Increases of uric acid and thiols levels were observed after ECC. Vitamin E levels were not modified. However after hemodilution correction a significant decrease of vitamin C level was noted in 24 h post-ECC samples as compared to per-ECC sample. Whatever the way of measurement, vitamin C levels decreased suggesting the occurrence of ECC induced-oxidative stress. Concerning RBC, in the absence of AAPH, extracellular potassium remained unchanged between pre-, per- and 24 h post-ECC. AAPH induced a significant increase in extracellular potassium and carbonyls levels of RBC membranes, which was not modified by ECC. These results suggest the absence of alterations of RBC membrane during ECC despite the occurrence of disturbances in PAS. Such protection is of particular importance in a cell engaged in the transport of oxygen and suggests that RBC are equipped with mechanisms affording a protection against free radicals.

Lidocaine has better antioxidant potential than ropivacaine and bupivacaine: in vitro comparison in a model of human erythrocytes submitted to an oxidative stress.

BACKGROUND: Local anesthetic agents may exert antioxidant properties in various models. The aim of this work was to compare the antioxidant properties of lidocaine, bupivacaine and ropivacaine using an in vitro model of human erythrocytes submitted to an oxidative stress. METHODS: Blood was obtained from healthy volunteers. After separation, erythrocytes were suspended in phosphate buffer. Oxidative stress was induced by incubation with 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). (1) Effects of four different concentrations (50, 100, 300 and 600 microg ml(-1)) of lidocaine, bupivacaine and ropivacaine were studied in absence or presence of AAPH (20 mM). Potassium efflux was assessed by flame photometry. (2) Effects of 50 and 600 microg ml(-1) of lidocaine, bupivacaine and ropivacaine on AAPH (50 mM) induced hemolysis were also studied. (3) The oxygen radical absorbing capacity of lidocaine, bupivacaine and ropivacaine at the four concentrations was evaluated by the analysis of the allophycocyanin fluorescence. RESULTS: In absence of AAPH, neither extracellular potassium nor hemolysis was noted. AAPH (20 mM) induced a significant increase in extracellular potassium that was reduced by all local anesthetic agents, with greater effects for lidocaine. AAPH-induced hemolysis was significantly decreased by all the local anesthetic agents at higher concentration, but only by lidocaine at 50 microg ml(-1). Finally, none of the local anesthetic agents modified the allophycocyanin fluorescence. CONCLUSION: In this model, lidocaine was proved more effective than bupivacaine and ropivacaine in protecting human erythrocytes submitted to an oxidative challenge. This was not due to a free radical scavenging effect.

Effects of various osmolarity on human red blood cells in terms of potassium efflux and hemolysis induced by free radicals.

OBJECTIVES: To study in an in vitro model of erythrocytes exposed to free radicals the effects of hyperosmolarity and hypoosmolarity on the induced potassium efflux and hemolysis. DESIGN SETTING: Erythrocytes were separated from plasma and suspended in 3 different phosphate buffer solutions (HYPO, ISO, and HYPER) containing, respectively, 100, 150 and 200 mmol/l of Na. Free radicals were generated from 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). Potassium efflux (flame photometry) and hemolysis (Drabkin method) were measured. Measurements were expressed in % (versus total) and area under % versus time curves were calculated (% min). An ANOVA was used for statistical analysis. RESULTS: In presence of AAPH, hemolysis was significantly greater in HYPO (732.45+/-40% min) and lower in HYPER (578.97+/-15% min) as compared to ISO (608.30+/-42% min). Potassium efflux was significantly increased in HYPER (7508.04+/-85% min) as compared with HYPO (5308.74+/-62% min) and ISO (6561.39+/-62% min). DISCUSSION: Our data suggest that hyperosmolarity increases the resistance of the red blood cells when exposed to free radicals as shown by the decreased hemolysis. In such conditions, the potassium efflux analysis appears to be inappropriate to evaluate the free radical effects on erythrocytes.

The protective effects of lidocaine on human erythrocytes stored for seven days at 04 degrees C.

Erythrocyte storage may result in cell damage due to an alteration of membrane integrity, which results in potassium efflux and hemolysis. Lidocaine has been shown to protect erythrocytes from oxidative stress by a possible membrane effect. We conducted this study to examine the effects of lidocaine on human erythrocyte storage. Erythrocytes were kept for seven days at 04 degrees C in the absence or in presence of plasma, and of lidocaine at 36.9 and 221.6 microM. Cell damage was assessed by measuring potassium efflux in the supernatant after seven days, and studying potassium efflux and hemolysis induced by oxidative stress. As expected, erythrocyte storage in the presence of plasma was less deleterious. Lidocaine decreased potassium efflux after 7 days' storage. Resistance toward oxidative stress was greater when the erythrocytes had been kept in the presence of plasma. Considering that lidocaine is widely used in various clinical situations, this data may be of clinical relevance.

Systemic free radical activation is a major event involved in myocardial oxidative stress related to cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) can induce deleterious effects that could be triggered in part by radical oxygen species; however, their involvement in the course of surgery has been elusive. The aim of this study was to evaluate the time course and origin of radical oxygen species release, myocardial or not, in patients undergoing coronary artery surgery involving CPB. METHODS: Blood samples were taken from periphery and coronary sinus of patients during CPB, and oxidative stress was evaluated by direct and indirect approaches. Direct detection of alkyl and alkoxyl radicals was assessed by electron spin resonance spectroscopy associated with the spin-trapping technique using alpha-phenyl-N-tert-butylnitrone. RESULTS: The authors showed that the spin adduct concentration was not influenced by anesthesia and pre-CPB surgery. A rapid systemic increase of plasma spin adduct concentration occurred after starting CPB, and it stayed at a high concentration until the end of CPB. At the beginning of reperfusion period, radical oxygen species release was accelerated in the coronary sinus; however, it was not significant. A positive correlation was found between alpha-phenyl-N-tert-butylnitrone adduct concentrations and (1) the duration of CPB and (2) concentration of postoperative creatine phosphokinase of muscle band (CPK MB). Plasma vitamin E and C, ascorbyl radical, uric acid, thiol, plasma antioxidant status, and thiobarbituric acid reacting substances were also measured but did not give relevant indications, except for uric acid, which seemed to be consumed by the heart during reperfusion. CONCLUSION: The results indicate that a systemic production of free radicals occurs during CPB that may overwhelm the production related to reperfusion of the ischemic heart. This systemic oxidative stress is likely to participate in secondary myocardial damage.