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PubMed1998

The determination of benzalkonium chloride in eye-drops by difference spectrophotometry.

A direct, extraction-free spectrophotometric method was developed for the determination of benzalkonium chloride (BAC) in various eye-drops. The procedure is based on ion-pair formation between BAC and 2',4',5',7'-tetrabromofluorescein (eosin-Y) which decreases the absorbance and induces a bathochromic shift of the maximum in the eosin-Y spectrum. The effects of pH, excess of reagent and ionic strength on the ion-pair formation have been studied in detail. At pH 4.40 and 9.62, the working curve is linear in the 1.98 x 10(-6) to 2.40 x 10(-5) M (0.7-8.5 microg cm[-3]) concentration range; however, the sensitivity drops to about one third in the basic solution. At pH 4.40, the analytical signal is stable for more than 60 min, while at pH 9.62 the signal changes in time and reaches the maximum value 3 min after mixing the reagent and the sample. When the active substance is beta-5-isopropyl-2'deoxyuridine and the sample contains typical additives, the reproducibility of the analytical signal at pH 4.40 is R.S.D. = 2.36% (n = 81). In the case of such samples, the linearity of the method is somewhat dependent on the composition, but generally acceptable at the 50-150% concentration levels. Eye-drops containing tobramycin, an aminoglycoside-type antibiotic, as the active substance were analyzed at pH 9.62. This was necessary to avoid strong interference from the analyte in acidic solution. In this case the linearity of the method is limited to a narrower concentration range; however, the recovery is still acceptable at the 100% level.

Benzalkonium Compounds↗
PubMed1998

Lidocaine and benzalkonium analysis and titration in drugs using new ISFET devices.

Two new ISFETs recently developed by us have now been applied to some pharmaceutical determinations in real matrices; the first device, responsive to cationic surfactants, was employed in the determination of benzalkonium chloride contained in two different disinfectant solutions and in three types of commercial collyrium; the second device, responsive to cocaine hydrochloride, showed an appreciable response also to lidocaine hydrochloride and was used in the determination of lidocaine hydrochloride contained in some injectable antibiotics. The repeatability and accuracy of measurements performed in the analysis of these pharmaceutical matrices using new solid state sensors were evaluated. A further aspect of the research involved the use of two sensors to record complete titration curves for the determination of benzalkonium chloride, cocaine hydrochloride and lidocaine hydrochloride, respectively. Applications to real matrices were also performed by analysing by titration pharmaceutical formulations containing benzalkonium chloride, or lidocaine hydrochloride and an illicit powder containing cocaine hydrochloride and sugars.

Benzalkonium Compounds↗
PubMed2001

[Acute hemolysis following perioperative blood salvage in chldren operated for the surgical correction of craniosynostosis].

We report the case of a 6-month-old child, who suffered from acute haemolysis following transfusion of salvaged blood. This complication, of favourable outcome, was related to the accidental aspiration of benzalkonium chloride into the cell saver. This case emphasizes that any adjunction of antiseptic solution is contraindicated during blood saving. The use of a cell saver must be associated with written protocols, describing clearly the contraindications, precautions of use, and the different steps of use of this method of autologous blood transfusion.

Acute Disease↗
PubMed2001

Toxic endothelial cell destruction from intraocular benzalkonium chloride.

PURPOSE: To present a cluster of cases of corneal edema occurring after phacoemulsification. SETTING: Regional hospital in rural Australia. METHODS: This retrospective case series comprised 12 patients from a single surgical list comprising 19 cases who developed persistent corneal edema after having phacoemulsification. RESULTS: After 6 months, the edema improved in 1 patient. Inadvertent intraocular use of balanced salt solution (BSS) preserved with benzalkonium chloride was found to be the cause of this serious complication. CONCLUSIONS: Benzalkonium chloride in concentrations commonly used extraocularly is highly toxic to the corneal endothelium. Every possible safeguard should be taken to prevent inadvertent intraocular use of such preserved solutions.

Acetates↗
PubMed2002

In vitro microbicidal activity of W/O/W multiple emulsion for vaginal administration.

The microbicidal activity of a W/O/W multiple emulsion destined for vaginal application, containing lactic acid in the internal aqueous phase, octadecylamine (ODA) in the oily phase and benzalkonium chloride (CBZ) in the external aqueous phase was evaluated against three microbial strains: Escherichia coli, Staphylococcus aureus and Candida albicans. The results were different depending on the procedure used. Interpretable results were obtained if only a gentle agitation was used just after the introduction of the microbial suspension to the product. This suggested that vigorous agitation lead to a variable fraction of CBZ or ODA entrapped in the micelles of ethylene and propylene oxide copolymer (COE).

Administration, Intravaginal↗
PubMed1999

[Antiseptic treatment of the umbilical cord in newborns: survey and recommendations].

AIM: To determine whether umbilical cord care of the neonate is in accordance with the guidelines of antiseptic treatment at this age of life. MATERIAL AND METHODS: A survey was conducted during the 3rd trimester of 1996 in 57 maternity units and departments of neonatalogy in the region of Provence-Alpes-Côte d'Azur (south of France). A questionnaire was sent to the head of each unit asking the modalities of disinfection of the umbilical cord. RESULTS: Fifty units answered the questionnaire. Six different groups of antiseptic products were used, corresponding to 17 distinct commercial preparations. The simultaneous association of several products (two or three) was done in 70% of cases. Eosin was the most frequently used (60%), in association with 25 units. Alcohol was used in 28 centers (56%). It was associated 22 times. Chlorhexidine was used in 16 units (32%), twice alone, and with another topic 14 times. The commercial association chlorhexidine-benzalkonium chloride (Biseptine) was reported seven times (six times in association with another topical treatment). Ektogan (a powder of Zn and Mg peroxide and Zn oxide) was used in ten centers, always in association. Hexamidine was used in four units, once in association. Silver nitrate, Milian solution, iodinated alcohol, and povidone iodine were respectively used once. CONCLUSION: This survey shows that a great variety of umbilical cord care modalities is used in this region, and that the recommendations for antiseptic treatment in young babies, are not always respected. According to these, eosin, ethanol, Ektogan and iodine should not be used for this purpose. Although chlorhexidine has been proven to be the most suitable disinfectant, it comes only in third place, used in association in 95% of the cases. Several studies in neonates have shown that it is well tolerated and efficient even if it delays cord separation. This study should lead to interdisciplinary consensual guidelines for umbilical cord care.

Anti-Infective Agents, Local↗
PubMed2001

Optical waveguide lightmode spectroscopy as a new method to study adhesion of anchorage-dependent cells as an indicator of metabolic state.

Optical Waveguide Lightmode Spectroscopy (OWLS) is based on measurements of the effective refractive index of a thin layer above the waveguide. Its potential as a whole-cell biosensor was demonstrated recently monitoring adhesion and spreading of Baby Hamster Kidney (BHK) cells on-line. In this work the OWLS is shown to be a promising tool to study the adhesion, morphology and metabolic state of fibroblasts in real time. A new design of the measuring chamber allowed simultaneous observation by phase-contrast microscopy and made the adsorbed cell density controllable and reproducible. The OWLS signal correlated quantitatively with the contact-area between the fibroblasts and the waveguide. The OWLS signals for adhesion and spreading of three different fibroblast cell lines were in good agreement with their morphology identified by phase-contrast microscopy. The cell adhesion and cell shape changes were examined in three scenarios: (a) serum-induced spreading of the surface attached fibroblasts was followed until it was completed, and the OWLS signal remained constant for over 12 h; (b) the fully spread cells were exposed to the microtubuli-disrupting colchicine and a decrease of the OWLS signal was monitored; (c) in a similar experiment with benzalkonium chloride, a strong skin irritant, a concentration-dependent response of the signal was found. The results show the strength of the OWLS method for monitoring the adhesion behavior of anchorage-dependent cells such as fibroblasts. It has a great potential as a whole-cell biosensor for high throughput screening in toxicology.

3T3 Cells↗
PubMed1997

[Quantitative bacteriological evaluation of a method for skin disinfection in blood donors].

Skin disinfection at the site of venipuncture is a critical point in every blood transfusion collection procedure, as it contributes to ensure the bacterial safety of transfusion. Quantitative and qualitative analysis of bacteria present in the antecubital fossae before and after skin disinfection may be one method of assessing the anti-bacterial efficiency of disinfection. Swab culture systems and contact plates are the two techniques usually employed for this purpose. A washing and swabbing technique was used to quantify bacteria before and skin disinfection of the antecubital fossae in blood donors. This contra-placebo study was carried out on 32 donors, each of whom served as his own control, with a random choice of test arm and opposing control arm. Bacterial counts were determined in the antecubital fossae without skin disinfection (control, n = 32) and after a 3 step skin preparation procedure (cleaning, wiping, disinfection) using placebo (distilled water, n = 16) or an antiseptic product (mixture of chlorexidine, benzalkonium chloride and benzylic alcohol, n = 16). The absence of a statistical difference in bacterial counts between the right and left antecubital fossae without disinfection was controlled in a preliminary study of 20 subjects. Mean bacterial counts were 25,000/cm2 and 27,400/cm2 respectively for aerobic and anaerobic bacteria before disinfection, with a wide variation in results between individuals. When using placebo, preparation of the venipuncture site by the 3 step method (cleaning, wiping, disinfection) resulted in a non significant mean reduction of 0.56 log in aerobic and anaerobic bacteria. Using the antiseptic product, the same method resulted in a significant mean reduction of 1.8 and 1.7 log respectively in aerobic (p = 0.015) and anaerobic flora (p = 0.005). On an average, 2,750 aerobic bacteria/cm2 and 2,910 anaerobic bacteria/cm2 remained after disinfection, while qualitative analysis showed that disinfection suppressed the transitory flora in all cases but left part of the resident flora in 12/16 cases. These findings are comparable to those of other studies carried out to evaluate this kind of technique for the disinfection of operation sites. In comparison with other techniques classically employed for this type of evaluation (swab systems or contact plates), the method used in this study was the advantage of allowing the quantification of the reduction in bacteria. Hence this method could be employed for comparative assessment of skin disinfection techniques with the aim of improving their anti-bacterial efficiency and could also make possible the definition of a minimum bacterial count (resident flora) to be obtained in all cases after disinfection.

Adult↗
PubMed2006

Effects of antiglaucoma drugs on cellular proliferation in cultured human corneal keratocytes.

The purpose of this study was to investigate the effects of various antiglaucoma drugs, including timolol, betaxolol, carteolol, levobunolol, dipivefrin, and pilocarpine, on cellular proliferation in cultured human corneal keratocytes. Human corneal keratocytes were cultured with RPMI-1640 medium containing 10% fetal bovine serum. Antiglaucoma drugs were prepared from original concentrations to dilutions of 1/10, 1/100, and 1/1,000. After exposure to drugs for 100 minutes, cellular proliferation was estimated by [3H]thymidine uptake methodology. It was found that cellular proliferation in corneal keratocytes was inhibited by only a 1/10 dilution of various drugs including timolol, betaxolol, carteolol, levobunolol, dipivefrin, and pilocarpine. The [3H]thymidine uptake values were significantly inhibited to 63%, 18%, 87%, 68%, 55%, and 67% by a 1/10 dilution of the above drugs. However, the cellular proliferation was also significantly suppressed by 0.01 mg/mL of benzalkonium chloride preservative. It is shown that the inhibition of cellular proliferation by high concentrations of antiglaucoma drugs may result from the benzalkonium chloride preservative contained in these drugs.

Benzalkonium Compounds↗
PubMed2006

Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes.

Liposomes containing phosphatidylcholine (PC) and cholesterol (Chol) were applied to the enhancer for firefly bioluminescence (BL) assay for ATP in the presence of cationic surfactants using as an extractant for the release of ATP from living cells. Benzalkonium chloride (BAC) was used as an ATP extractant. However, BAC seriously inhibited the activity of luciferase, thus resulting in the remarkable decrease in the sensitivity of the BL assay for ATP. On the other hand, we found that BAC was associated with liposomes to form cationic liposomes containing BAC. The association rate of BAC with liposomes was faster than that of BAC with luciferase. As a result, the inhibitory effect of BAC on luciferase was eliminated in the presence of liposomes. In addition, cationic liposomes thus formed enhanced BL emission. BL measurement conditions were optimized in terms of liposome charge type, liposome size, and total concentration of PC and Chol. ATP can be sensitively determined without dilution of analytical samples by using liposomes. The detection limit of ATP with and without liposomes was 100 amol and 25 fmol in aqueous ATP standard solutions containing 0.06% BAC, respectively. The method was applied to the determination of ATP in Escherichia coli extracts. The BL intensity was linear from 4 x 10(4) to 1 x 10(7) cells mL(-1) in the absence of liposomes. On the other hand, the BL intensity was linear from 4 x 10(3) to 4 x 10(6) cells mL(-1) in the presence of liposomes. The detection limit of ATP in E. coli extracts was improved by a factor of 10 via use of liposomes.

Adenosine Triphosphate↗
PubMed2001

Evidence for simultaneous binding of dissimilar substrates by the Escherichia coli multidrug transporter MdfA.

The mechanism by which multidrug transporters interact with structurally unrelated substrates remains enigmatic. Based on transport competition experiments, photoaffinity labeling, and effects on enzymatic activities, it was proposed in the past that multidrug transporters can interact simultaneously with a number of dissimilar substrate molecules. To study this phenomenon, we applied a direct binding approach and transport assays using the Escherichia coli multidrug transporter MdfA, which exports both positively charged (e.g., tetraphenylphosphonium, TPP(+)), zwitterionic (e.g., ciprofloxacin), and neutral (e.g., chloramphenicol) drugs. The interaction of MdfA with various substrates was examined by direct binding assays with the purified transporter. The immobilized MdfA binds TPP(+) in a specific manner, and all the tested positively charged substrates inhibit TPP(+) binding. Surprisingly, although TPP(+) binding is not affected by zwitterionic substrates, the neutral substrate chloramphenicol stimulates TPP(+) binding by enhancing its affinity to MdfA. In contrast, transport competition assays show inhibition of TPP(+) transport by chloramphenicol. We suggest that MdfA binds TPP(+) and chloramphenicol simultaneously to distinct but interacting binding sites, and the interaction between these two substrates during transport is discussed.

Bacterial Proteins↗
PubMed2004

Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA.

According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition. To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized. Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge. Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism. Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets.

Amino Acid Sequence↗