Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Enzyme immunoassay of human plasma fibronectin in malignant, collagen and liver diseases.

A competitive enzyme immunoassay for the measurement of human plasma fibronectin (PFN) was developed, using polystyrene beads as a solid phase and glucose oxidase as a marker enzyme. This method gave a reproducible standard curve in the range of 0.5-50 micrograms/ml. The mean PFN level in normal persons was 345 micrograms/ml (standard deviation 68.4 micrograms/ml, n = 30). The PFN levels in patients with malignant, collagen and liver diseases were also measured. The mean PFN level in patients with collagen disease was significantly higher than that in normal persons, whereas the mean PFN levels in patients with malignant and liver diseases were not significantly different.

A new treatment of aneurysmal bone cyst by endoscopic curettage without bone grafting.

Curettage and bone grafting are the accepted methods of treatment of aneurysmal bone cysts. Unfortunately, recurrence is common. We treated 4 patients with atypical aneurysmal bone cysts that lacked aneurysmal dilatation by endoscopic curettage without bone grafting. New bone formation and remodeling were observed in all patients. In the patients in whom the follow-up was longer than 30 months, there was no evidence of recurrence. Endoscopic curettage without bone grafting is a simple and effective treatment for aneurysmal bone cysts.

[Anesthetic management of a patient with pemphigoid for prosthetic replacement after femoral neck fracture].

We report an 83 year-old male with pemphigoid who underwent replacement of a femoral neck prosthesis following femoral neck fracture. Problems associated with anesthetic management of patients with vesiculobullous diseases such as pemphigoid include fluid, electrolyte and protein loss, and secondary infection because of the fragility of the skin and mucous membranes. Avoidance of trauma to the skin and mucous membranes is therefore important. Intubation of the trachea may be hazardous because of the possibility of airway obstruction resulting from oropharyngeal and laryngeal bullae. The patient was successfully anesthetized with spinal anesthesia using 2.5 ml of tetracaine in a 0.4% solution of 0.45% NaCl. An evacutable mattress was used to protect the patient from pressure points in the lateral position, and to minimize the hazards from pressure by spreading the weight. Surgery proceeded uneventfully and the postoperative period was uncomplicated.

Temperature dependence of retention in reversed-phase liquid chromatography on a porous acrylic support.

The solute retention mechanism of reversed-phase liquid chromatography is examined by using newly synthesized hydrophilic porous copolymer beads as a stationary phase. It is shown that when water is used as an eluent, the retention time for solutes such as alcohols, ketones and polyethylene glycols increases with temperature. Adsorption behaviour of polyethylene glycol onto the copolymer beads shows that the adsorption also increases with temperature. These observations are explained on the basis of the hypothesis that hydrophobic interaction plays a vital role in solute retention.

Electrophysiological studies on the evaluation of absorption enhancers in Caco-2 cells using a microelectrode technique.

We developed a microelectrode technique to characterize the electrophysiological properties in Caco-2 cells, and used it to determine the mechanisms of absorption enhancers. The action of absorption enhancers on the apical membrane of Caco-2 cells was estimated by measuring the apical membrane potential (Vm) with the microelectrode. The Vm value of Caco-2 cells in Hanks' balanced salt solution containing 0.5 mM K+ was 18.9+/-0.8 mV (n=217), and the apical membrane resistance was 49.4+/-1.1 MOhms (n=160). In the electrophysiological study with absorption enhancers, laurylmaltoside (LM) markedly decreased the Vm value, while sodium glycocholate (NaGC) moderately reduced this value, and EDTA did not affect the value. These findings might be associated with their action sites, plasma membrane or tight junction in Caco-2 cell monolayers. In influx and transport studies with these absorption enhancers, LM enhanced the influx of furosemide, which is transported via both the transcellular and paracellular routes into Caco-2 cells, and enhanced its transport to the basolateral side of Caco-2 monolayers more than that of 5(6)-carboxyfluorescein (CF), a paracellular marker. EDTA did not increase the influx of furosemide, and enhanced the transport of furosemide and CF across Caco-2 cell monolayers to the same extent. In contrast, NaGC only slightly increased the influx of furosemide and did not enhance the transport of either furosemide or CF across the Caco-2 monolayers in this study. These findings were well correlated with the effects of these absorption enhancers on the electrophysiological parameters. Therefore, the microelectrode technique might be useful for evaluating the action of absorption enhancers in the plasma membrane at an intact cell level.

Rapid measurement of oestradiol and oestriol by high-performance liquid chromatography after automatic pretreatment.

A fluorometric liquid chromatographic method was developed for measurements of unconjugated oestradiol and oestriol in the serum of pregnant women. The serum samples are injected directly into the apparatus and pass to a pretreatment column, where oestrogens are adsorbed while hydrophilic components such as proteins and carbohydrates are not. The oestrogens then pass into a separation column containing a new type of polymer gel. The mobile phase consists of an acetonitrile-water mixture, and separation is achieved by a reversed-phase mechanism. The eluent is monitored for fluorescence. Data on the reproducibility and recovery by this method and the correlation of values with those obtained by radioimmunoassay are reported. Results on the increases of oestradiol and oestriol in the serum during pregnancy are also reported.

Popliteal pseudoaneurysm simulating soft-tissue sarcoma: complication of osteochondroma resection.

Pseudoaneurysm complicating osteochondroma is extremely rare. We describe a pseudoaneurysm in the popliteal fossa of a 21-year-old man, occurring 5 years after apparently incomplete resection of an osteochondroma at that site. We found magnetic resonance imaging and magnetic resonance angiography to be of great diagnostic value. Resection of the pseudoaneurysm and anastomosis of the popliteal artery were successful, with no recurrence detected for almost 7 years since the operation.

Results of surgery and radio-hyperthermo-chemotherapy for patients with soft-tissue sarcoma.

BACKGROUND: Between 1990 and 1999, we performed radio-hyperthermo-chemotherapy (RHC) in 44 patients with high-grade soft-tissue sarcomas of the limbs. METHODS: Radiotherapy involved the delivery of radiation at a dose of 2 Gy once daily on 16 days, to give a total dose of 32 Gy. Hyperthermia was conducted once a week, with a total of five sessions. Chemotherapy was performed by implanting a reservoir and administering cisplatin (3 mg/kg) three times, and pinorubin (an adriamycin derivative; 1 mg/kg) twice by intra-arterial infusion, at weekly intervals. These drugs were administered alternately during hyperthermia sessions. RESULTS: Tumor shrinkage was observed in 98% (43/44) of the patients. Of the 36 patients with M0 tumors, 30 were disease-free at final follow-up, 2 had no evidence of disease, 1 was alive with disease, and 3 had died of the disease. Amputation was required only in the first patient, and the affected limb was preserved in the other 43 patients. The surgical margin was wide in 9 patients and marginal in 29 patients, and intralesional excision was performed in 5 patients. There was recurrence in only 1 of the 44 patients. CONCLUSION: RHC is currently the most potent and relatively safe treatment method for high-grade soft-tissue sarcomas that is available clinically.

A highly sensitive determination of individual serum bile acids using high-performance liquid chromatography with immobilized enzyme.

We have developed a highly sensitive method for the simultaneous determination of individual bile acids in serum using high-performance liquid chromatography (HPLC) combined with immobilized 3 alpha-hydroxysteroid dehydrogenase. Both the HPLC column and the immobilized-enzyme column are suitable for use with alkaline solutions necessary in working with this enzyme system. With this method we were able to determine simultaneously fifteen different serum bile acids.

Inhibition of vitronectin-mediated haptotaxis and haptoinvasion of MG-63 cells by domain 5 (D5(H)) of human high-molecular-weight kininogen and identification of a minimal amino acid sequence.

We found that human kinin-free high-molecular-weight kininogen (kf-HK) significantly inhibited vitronectin-mediated migration (haptotaxis) and invasive potentiation (haptoinvasion) of osteosarcoma (MG-63) cells but that HK, LK, the common heavy chain of HK and LK, and the light chain (D6(H)) of HK had no inhibitory effect. Recombinant GST-D5(H) (histidine-rich region of HK) obtained from Escherichia coli. (BL21) also inhibited both haptotaxis and haptoinvasion to about 30% of the control level in a dose-dependent manner. These findings suggest that a specific region of D5(H) is responsible for the inhibition of cell haptotaxis and haptoinvasion. Among the seven synthetic peptides covering D5(H), peptide H(479)KHGHGHGKHKNKGK(493) (P-5) inhibited both haptotaxis and haptoinvasion in a dose-dependent manner, suggesting that P-5 could possibly be utilized to prevent primary and secondary metastases of tumor cells.