Protein microdeposition using a conventional ink-jet printer.

Many recent bioanalytical systems based on immunologic and hybridization reactions in a mono- or bidimensional microarray format require technology that can produce arrays of spots containing biospecific molecules. Some microarray deposition instruments are commercially available, and other devices have been described in recent papers. We describe a system obtained by adapting a commercial ink-jet printer and used to produce mono- and bidimensional arrays of spots containing horseradish peroxidase on cellulose paper. In a few minutes, it was possible to obtain bidimensional arrays containing several thousands of spots with a diameter as low as 0.2 mm, with each of which requiring only a few nanoliters of the enzyme deposition solution. The quantity of enzyme in each spot was evaluated with a chemiluminescent reaction and a charge-coupled device-based, low-light imaging luminograph. The chemiluminescence measurements revealed that the reproducibility of the enzyme deposition was satisfactory for analytical purposes, with the variation coefficients being lower than 10% in almost all cases.

Co-localization of two different viral genomes in the same sample by double-chemiluminescence in situ hybridization.

A double-chemiluminescence in situ hybridization has been developed that combines the advantages of chemiluminescence with the detection of two different viral DNAs, i.e., herpes simplex virus (HSV) DNA and cytomegalovirus (CMV) DNA, in infected cells in the same specimen. For the simultaneous detection of these two different viral DNAs, we used a biotinylated HSV DNA probe, which can be visualized by a streptavidin-horseradish peroxidase (HRP) complex amplified with biotinyl tyramide. This probe was followed by the use of a luminol-based chemiluminescent substrate for HRP and a digoxigenin-labeled CMV DNA probe visualized by antidigoxigenin Fab fragments conjugated with alkaline phosphatase (AP). This is followed by the detection with a dioxetane phosphate derivate as chemiluminescent substrate for AP. Since the final product of both chemiluminescent reactions was light emission, sequential images for the two hybridizations were taken and analyzed using a high-performance luminograph connected to an optical microscope and to a personal computer for image analysis. Positive signals for the presence of both HSV DNA and CMV DNA were noticed in infected cells in the same specimen with a sharp localization, absence of cross reactions and absence of background.

Experimental evaluation of a model for predicting micellar composition and concentration of monomeric species in bile salt binary mixtures.

The critical micellar concentration (cmc) values of some mixed systems containing two bile salts were determined by a maximum pressure bubble method and compared with those derived from a theoretical model developed for nonionic surfactants to assess the applicability of this model to such systems. Some assumptions on which the presumed validity of this model was based are discussed. The following binary mixtures were investigated: sodium chenodeoxycholate with cholate, ursocholate and ursodeoxycholate, either unconjugated or conjugated with taurine and glycine at different mole fractions (0, 0.25, 0.5, 0.75, 1) in 0.15 M NaCl. For these mixtures, experimentally determined data were in good agreement with values predicted by the theoretical model: both the cmc and the surface tension at this concentration of the mixtures were intermediate between those of the two pure bile salts; also, as the total bile salt concentration increased, the mixed micelles became enriched with the bile salt having the highest cmc, whereas the total monomer activity, determined by a potentiometric method employing a bile salt-selective electrode, increased only slightly. To test this model in an in vitro system, surface tension was also measured in ox bile samples that were enriched by 50% with sodium ursodeoxycholate, chenodeoxycholate, or their taurine amidates. The cmc and the surface tension at this concentration of the artificial bile increased when enriched with a bile salt with a cmc higher than that of endogenous salts (e.g. ursodeoxycholate versus taurocholate), whereas the reverse occurred for mixtures enriched with a bile salt with a lower cmc, such as chenodeoxycholate.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioavailability of a new ketoprofen formulation for once-daily oral administration.

A new sustained-release formulation (sustained release Ibifen) that gradually releases ketoprofen within 24 h and ensures therapeutic plasma concentration for the entire period has been developed. It consists of tableted pH-dependent barrier film-coated ketoprofen granules and was administered at a single dose of 200 mg to 12 volunteers. Ketoprofen plasma profiles were compared with: (1) administration of Orudis retard 200 capsule (200 mg); (2) two 12-h doses of prompt release Ibifen capsules (100 mg). In vitro dissolution kinetics and ketoprofen plasma levels were measured by HPLC. Sustained release Ibifen dissolution rate was constant for 10 h, whereas Orudis retard 200 dissolution profile presented one higher slope (0-6 h) and a lower one (6-12 h). Both formulations showed a delayed kinetics with respect to prompt release Ibifen. After sustained release Ibifen administration, ketoprofen plasma peak, reached within 2 h, remained practically constant for at least 12 h (average 4 microg/ml), which is higher than therapeutic levels. Differently, Orudis retard 200 produced a delayed, higher C(max) (5.91+/-0.66 vs. 4.51+/-0.65 microg/ml; P<0.01) and disappeared more quickly. In conclusion, sustained release Ibifen can ensure therapeutic ketoprofen plasma levels for the entire 24 h period, avoiding plasma concentration spikes, with bioavailability similar to other ketoprofen preparations.

Chemiluminescence imaging in bioanalysis.

The development, analytical performance and applications of chemiluminescence imaging as a tool for quantitative analyte localization in target biological specimens are described. The detection of acetylcholinesterase activity both in array format and on a target surface are described. A proposed application of the method is a 384 well microtiter format assay for high throughput screening of acetylcholinesterase inhibitors such as tacrine, a drug widely used in the treatment of Alzheimer's disease, and two recently developed analogues. The chemiluminescent system in conjunction with optical microscopy allowed localization of acetylcholinesterase in brain tissue sections. We also describe the chemiluminescent immunohistochemical localization of interleukin 8 in Helicobacter pylori infected gastric mucosa cryosections and an in situ hybridization assay for the detection of herpes simplex virus DNA in single cells.

Influence of cimetidine in low doses (less than ED50) on prostanoid production by human gastric mucosa in vitro.

Cimetidine has been defined as a cytoprotective agent and numerous studies have reported that it is able to influence prostaglandin production as well as mechanisms which protect the surface epithelium of the gastric mucosa. However, results have been contradictory and high drug concentrations contrasting the cytoprotection concept have been utilized. The present study tried to evaluate whether low concentrations (less than ED50) of cimetidine in vitro are able to modify prostanoids produced by gastric mucosa fragments. Thirteen patients without histological lesions were examined. Five mucosal biopsy specimens were obtained from the antrum in seven patients and from the body in another six patients. One biopsy was utilized for histological examination, while the remaining four specimens were incubated in the absence or in the presence of 5, 50 and 500 ng/ml of cimetidine, respectively. Concentrations of PGE2, 6-oxo-PGF1 alpha, PGF2 alpha and TXB2 in the incubate were determined by radioimmunoassay. Cimetidine at 50 ng/ml increased PGE2 production at the antrum level while 500 ng/ml of cimetidine increased PGF 2 alpha and TXB2 production at the body level. Furthermore, the drug dose was directly related to PGE2 production (at the antrum level) and TXB2 (at the body level). The differences in prostanoid production between the antrum and body could be due to the different cell composition of the two anatomical areas, and suggest that the effects of cimetidine are mediated through binding to H2-receptors. The authors conclude that cimetidine in low doses stimulates the net production of some but not all prostanoids, the observed effects varying with anatomical site.

Pharmacokinetics and bioavailability of four modified-release ursodeoxycholic acid preparations for once-a-day administration.

Ursodeoxycholic acid (UDCA) is currently used for the treatment of cholestatic liver disease and for cholesterol gallstone dissolution. Various formulations have been designed to enhance its intestinal absorption or to improve patient compliance through once-a-day administration. The pharmacokinetics and bioavailability of four commercially available modified-release UDCA formulations (450 mg) were studied in 12 healthy subjects randomly receiving the four drugs under study. Serum samples were collected hourly for a 12-h period after administration and UDCA concentrations were measured using a specific enzyme immunoassay. For each formulation, Cmax, tmax, and the area under the curve (AUC) were determined and compared. Although all formulations were designed to provide sustained release, we observed different pharmacokinetics among the studied formulations. One of the formulations (sustained-release ursodeoxycholic acid Ratiopharm 450 mg tablets) showed high bioavailability, but failed to produce sustained release. In contrast, two other formulations (modified-release ursodeoxycholic acid Dorom 450 mg capsules and controlled-release Ursobil HT 450 mg capsules) provided sustained release, but did not offer efficient bioavailability. A fourth formulation (Ursilon retard 450 mg) exhibited gradual UDCA release lasting over 10 h, with efficient bioavailability, similar to that of conventional prompt-release formulations administered at the same dose. These data highlight the variability of commercially available sustained-release formulations. Manufacturers should be encouraged to provide drug kinetics and bioavailability data to further support the claimed pharmacokinetics.

Protection of the upper gastrointestinal mucosa: the role of antacids.

Experiments performed in animals and in healthy human subjects suggest that antacids increase prostaglandin synthesis and have a cytoprotective effect on gastroduodenal mucosa. To investigate this hypothesis, the ability was evaluated of an antacid containing an aluminium/magnesium hydroxide combination (Maalox TC) to modify prostanoid production at the gastric level in 28 patients with gastric antral ulcer of various sizes in different stages of activity with or without erosive gastritis. After the antacid treatment, a significant prostaglandin E2 reduction was observed, together with a significant 6-keto-prostaglandin F1 alpha increase, but there was no thromboxane B2 variation at antrum level, nor any significant modification of prostanoid production at body level. The decreased prostaglandin E2 levels, detected after treatment with the antacid combination, may be due to lesion improvement, decreased synthesis or increased catabolism by mucosal cells, to a drop in this prostaglandin production by inflammatory cells. As far as 6-keto-prostaglandin F1 alpha is concerned, the obtained data confirm the results reported by other authors in healthy human subjects. The increase of this prostaglandin could enhance blood flow, resulting in a protective effect.

Oxmetidine (SK&F 92994): pharmacokinetic study in patients with in patients with liver cirrhosis.

The pharmacokinetics of oxmetidine (SK&F 92994) were investigated in nine cirrhotic patients and compared with ten control subjects with gastroduodenal ulcers, but without any symptoms of hepatic pathology. On two separate occasions each patient received 200 mg oxmetidine as a single oral dose and 100 mg as a single intravenous dose. In the cirrhotics, the bioavailability of the oral dose and the plasma elimination half-life after both oral and intravenous administration were significantly higher than in the controls. Moreover, a positive correlation was found between the plasma elimination half-lives and the biochemical parameters of cholestasis. Such findings indicate that in severe liver disease and in cholestasis the accumulation of oxmetidine in the circulation may limit the use of this drug.

[Effects of tiropramide on gallbladder emptying in patients with a dyspeptic syndrome associated with biliary dyskinesia]. / Effetti della Tiropramide sullo svuotamento colecistico in portatori di sindrome dispeptica correlata a discinesia biliare.

Thirty patients with biliary dyskinesia were treated with 300 mg/t.i.d. of Tiropramide for 15 days. The statistical evaluation showed a significant reduction of symptoms, an increase (p less than 0.001) of volumes and an improvement (p less than 0.001) of gallbladder emptying.

Antacids in gastric ulcer treatment: evidence of cytoprotection.

Antacids are more effective than placebo, and their efficacy is comparable to that of H2-blockers in gastric ulcer healing even though the healing time is about 2 weeks longer than with H2-blockers. Some observations in animals and healthy subjects may indicate that antacids (particularly those containing aluminium hydroxide) have a protective effect on the gastroduodenal mucosa in that they increase the production of prostanoids and sulphydryl-containing compounds. We showed that 10 days' treatment with high-dose antacids (Maalox TC) is able to increase 6-keto-PGF1 alpha production from cultured biopsy specimens of patients with gastric ulcer. These data could constitute a further indication in the treatment of peptic disease.

Separation techniques for bile salts analysis.

The analysis of bile salts in biological samples has remained a difficult task, due to the complex nature of the salts and also to their low concentration in common sample fluids such as plasma and urine. Given their importance, the development of accurate and sensitive methods of instrumental analysis has been the subject of intensive research, and recent advances have eliminated or lessened some of the difficulties. Currently available techniques are the following: thin-layer chromatography, gas chromatography, high-performance liquid chromatography, supercritical fluid chromatography, gas chromatography-mass spectrometry and capillary electrophoresis. Liquid chromatography coupled with mass spectrometry (thermospray, fast atom bombardment, electrospray and ionspray), a method undergoing continuous improvement, is also being applied to bile salts analysis. In this paper, these various techniques, which differ greatly in specificity, accuracy and simplicity, are reviewed and discussed, in terms of analytical performance, applicability to a given sample fluid, major limitations, ability to identify uncommon bile salts, including unsaturated oxo derivatives, glucuronides, sulfates, glycosides and bile alcohols.

High-performance liquid chromatographic-electrospray mass spectrometric analysis of bile acids in biological fluids.

The present work describes the development of HPLC-mass spectrometric systems equipped with an electrospray interface for the quantitative analysis of bile acids. Good separation of free as well as glycine- and taurine-conjugated bile acids was achieved with a C18 reversed-phase column (3 microns particle size, 70 x 4.6 mm I.D.) employing methanol-15 mM ammonium acetate as the mobile phase for both isocratic and gradient mode, at a flow-rate of 0.3 ml/min. This system permits post-column splitting of the eluate for analysis by two different detectors: (1) electrospray-mass spectrometer with a flow-rate of 18 microliters/min; and (2) a complementary evaporative light scattering mass detector. When bile salts were ionized in the electrospray interface operating in the negative-ion mode, only [M-H]- molecular ions were generated; the detection limit was 15 pg injected for all bile acids studied. In the second system, a semi-micro pre-column splitting apparatus (Acurate, LC Packings) was utilized: with this device the flow-rate from the HPLC pump was reduced to 1.4 microliters/min and bile acids were separated with a micro-bore C18 column (3 microns particle size, 150 x 0.30 I.D.), using the same mobile phase as above. With this latter system, a head-column enrichment technique can be used: the amount injected can be increased from 60 to 200 nl, permitting an improvement in the detection limit to 5 pg injected. Application of the HPLC-electrospray-mass spectrometric method to bile and serum bile acid analysis is described; preliminary data on the ability of the first system to determine the 13C/12C isotope ratio in 13C-labeled bile acid enriched serum is also critically discussed.

Bio- and chemiluminescence in bioanalysis.

Analytical chemiluminescence and bioluminescence represent a versatile, ultrasensitive tool with a wide range of applications in diverse fields such as biotechnology, pharmacology, molecular biology, clinical and environmental chemistry. Enzyme activities and enzyme substrates and inhibitors can be efficiently determined when directly involved in luminescent reactions, and also when they take part in a reaction suitable for coupling to a final light-emitting reaction. Chemiluminescence detection has been exploited in the fields of flow-injection analysis and column-liquid chromatographic and capillary-electrophoretic separative systems, due to its high sensitivity when compared with colorimetric detection. It has widely been used as an indicator of reactive oxygen species formation in cells and whole organs, thus allowing the study of a number of pathophysiological conditions related to oxidative stress. Chemiluminescence represents a sensitive and rapid alternative to radioactivity as a detection principle in immunoassays for the determination of a wide range of molecules (hormones, food additives, environmental pollutants) and in filter membrane biospecific reactions (Southern, Northern, Western, dot blot) for the determination of nucleic acids and proteins. Chemiluminescence has also been used for the sensitive and specific localization and quantitation of target analytes in tissue sections and single cells by immunohistochemistry and in situ hybridization techniques. A relatively recent application regards the use of luminescent reporter genes for the development of bioassays based on genetically engineered microorganisms or mammalian cells able to emit visible light in response to specific inorganic and organic compounds. Finally, the high detectability and rapidity of bio- and chemiluminescent detection make it suitable for the development of microarray-based high throughput screening assays, in which simultaneous, multianalyte detection is performed on multiple samples.

Assessment of portal hypertension by endoscopic ultrasonography.

Endoscopic ultrasonography (EUS) was performed in 40 patients with portal hypertension (PH) and in 48 control subjects. The azygous, splenic, mesenteric, and portal veins were displayed in both groups. However, esophageal and gastric varices, periesophageal and perigastric collateral veins, and submucosal gastric venules were displayed only in patients with portal hypertension. EUS was inferior to endoscopy in detecting and grading esophageal varices (p less than 0.0005), but EUS was superior in the detection of varices in the fundus of the stomach (p less than 0.0005). Detection of periesophageal veins by EUS increased with increasing diameter of esophageal varices at endoscopy (57% in grade 1, 89% in grade 2, and 100% in grade 3), and there was a direct correlation between endoscopic grade and the diameter of the periesophageal collateral veins at EUS. The diameter of the azygous vein by EUS at its distal and proximal margins was significantly greater in patients with PH (p less than 0.001); the EUS diameter of the azygous vein was significantly larger with variceal grade 2 compared with grade 1 (p less than 0.02 and p less than 0.01, respectively). In portal hypertensive gastropathy, endoscopic and EUS detection were coincident. No correlation was found between the presence of portal hypertensive gastropathy, endoscopic grade of esophageal varices, and detection of gastric varices at EUS.

Chemiluminescent low-light imaging of biospecific reactions on macro- and microsamples using a videocamera-based luminograph.

The analytical performance of a low-light imaging luminograph for quantitative luminescence analysis was evaluated in terms of sensitivity, spatial resolution, accuracy, precision, and sample geometry, at the macrolevel and in combination with optical microscopy. The system allows for the detection of 400 amol of enzymes such as alkaline phosphatase and horseradish peroxidase using 1,2-dioxetanes and luminol/p-iodophenol or acridancarboxylate esters, respectively, as chemiluminescent substrates. Enzymatic activity and spatial distribution of nylon net immobilized-alkaline phosphatase was studied; the system permits the quantification of the immobilized enzyme with a spatial resolution as low as 1 µm. Other applications, such as the alkaline phosphatase localization in 8 µm intestinal mucosa cryosections, quantitative immunocytochemistry, and dot blot DNA hybridization reactions, were studied and optimized. The system was also employed for in situ hybridization assay of cytomegalovirus DNA in infected human fibroblasts. The presence of a viral genome was revealed with digoxigenin-labeled probes and alkaline phosphatase-labeled anti-digoxigenin antibody, using chemiluminescent substrate for this enzyme. The luminescent signal was intense and stable, and the probe was imaged and quantified within single cells with higher intensity in the nuclei, with a spatial resolution as low as 1 µm and very low background. The results show that this technique is an ultrasensitive and potent analytical tool to localize and quantify biomolecules at microscopic level, and it is suitable for many bioanalytical applications.