Evaluation of fast-acting bactericidal activity and substantivity of an antiseptic agent, olanexidine gluconate, using an ex vivo skin model.

PURPOSE: We assessed the fast-acting bactericidal activity and substantivity of olanexidine gluconate (OLG) to investigate its remaining bactericidal activity on the skin after rinsing and drying by using an ex vivo Yucatan micropig (YMP) skin model. METHODOLOGY: The fast-acting bactericidal activity was evaluated in pigskin models inoculated with methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, vancomycin-resistant Enterococcus faecalis (VRE), Acinetobacter baumannii, Corynebacterium minutissimum and Cutibacterium acnes. To evaluate substantivity, the YMP skin piece first had 1.5 % OLG, chlorhexidine gluconate (CHG) formulations or 10 % povidone-iodine (PVP-I) applied to it, and was then rinsed with distilled water, incubated for 4, 6, 8 or 12 h and inoculated with the test bacteria (MRSA, S. epidermidis and VRE). The viable bacteria remaining at 1 min of exposure of bacteria were counted to measure the quantity of antiseptic molecules retaining bactericidal activity. To determine the factors contributing to the substantivity, the stratum corneum (SC) of the YMP skin that had had OLG or CHG applied to it was exfoliated using a tape-stripping method and the amount of antiseptic was quantitated. RESULTS: OLG showed a fast-acting bactericidal activity that was similar to or stronger than that of CHG formulations up to a concentration of 1 % and PVP-I with a short exposure time of 30 s, and substantivity until 12 h after rinsing, whereas the other antiseptics hardly showed any substantivity. There was 2.8 times or more OLG in the SC than CHG. CONCLUSION: OLG has fast-acting activity and substantivity, which are required properties for an antiseptic, and is useful for preventing infections.

Effects of Parenteral Amino Acid Administration on the Postoperative Nutritional Status and Wound Healing of Protein-Malnourished Rats.

In Japan, parenteral nutrition (PN) solutions are frequently administered to patients in the postoperative short-term period. In these cases, amino acid-containing peripheral parenteral nutrition (PPN) solutions, amino acid-free maintenance solutions or combinations of the two are used. However, consensus regarding the most beneficial solution for these patients is lacking. Here, we examined the nutritional status and wound healing outcomes in protein-malnourished rats receiving postoperative administrations of PPN solution, maintenance solution or combinations of the two solutions. Protein malnutrition was induced in Sprague-Dawley rats by feeding an AIN-93G-based low-protein diet (5% casein) for 2 wk. After laparotomy, dorsal skin incision, and placement of a jugular vein catheter, the rats were divided into 3 groups. Each group was administered 113 kcal/kg/d, with group A receiving maintenance solutions without amino acid, group B receiving PPN with 1.5% amino acid, and group C receiving PPN with 3% amino acid. After 5 d post-operative administration, we measured the tensile strength of the wound area, skeletal muscle weights, and nutritional parameters. Significantly higher plasma nutritional parameters and gastrocnemius and extensor digitorum longus (EDL) muscle weights were observed in groups B and C than in group A. Group C exhibited significantly elevated tensile strength of the wound area along with up-regulation of type I collagen mRNA expression compared to group A. These findings demonstrate the nutritional status and wound healing benefits of short-term postoperative administration of PPN solutions containing amino acids in protein-malnourished rats.

Studies on neurosteroids XIX. Development and validation of liquid chromatography-tandem mass spectrometric method for determination of 5alpha-reduced pregnane-type neurosteroids in rat brain and serum.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method for the simultaneous determination of 5alpha-reduced pregnan-type neurosteroids, allopregnanolone (AP), epiallopregnanolone and 5alpha-dihydroprogesterone, in rat brain and serum has been developed and validated. The brain and serum steroids were extracted with methanol-acetic acid, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-positive ESI-MS-MS. The limits of quantitation (LOQ) for brain (0.25 ng/g tissue) and serum (0.25 ng/ml) assays using the derivatization-ESI-MS-MS method are 60-150-fold lower than the LOQs for their atmospheric pressure chemical ionization-MS method without derivatization. [17Alpha,21,21,21-2H4]-AP was used as an internal standard. This method allowed the reproducible and accurate quantification of the brain or serum neurosteroids using a 20 mg or 20 microl sample, respectively. That is, the intra- and inter-assay coefficients of variation were below 8.2 and 6.0%, respectively, and the % accuracy values were 98.5-103.0% for all the steroids in both the brain and serum. The application of the developed method to the analysis of changes in the brain and serum neurosteroid levels by immobilization stress and ethanol administration is also presented.

Studies on neurosteroids XXII. Liquid chromatography-tandem mass spectrometric method for profiling rat brain 3-oxo-4-ene-neuroactive steroids.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous determination of five 3-oxo-4-ene-neuroactive steroids, i.e. androstenedione, testosterone (T), progesterone (PROG), 20alpha-dihydroprogesterone and 20beta-dihydroprogesterone, in rat brain has been developed and validated. The brain steroids were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges and subjected to LC-ESI-MS/MS. The method does not require derivatization. Deuterium-labeled T and PROG were used as the internal standards, and quantification was based on the selected reaction monitoring mode. This method allowed the reproducible and accurate quantification of the brain neuroactive steroids using 100 mg of tissue; the intra- and inter-assay relative standard deviations were below 4.7 and 4.3%, respectively, and the accuracy values were 97.6-103.2% for all the steroids. The limits of quantitation were 0.1 ng/g tissue for all the steroids. The application of this developed method for the analysis of changes in the brain neuroactive steroid levels by immobilization stress is also presented.