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Biomedical subjects

C Arcelloni

Publications and source records attributed to C Arcelloni.

33 records · Page 2Linked to original sources
PubMed1997

Plasma mitomycin C concentrations determined by HPLC coupled to solid-phase extraction.

The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of local bladder hyperthermia (HT). In comparison with existing methods, this assay, characterized by relative simplicity and efficiency, resulted in the facilitation of performance with nondedicated instrumentation or nonspecialized staff. Purification from plasma matrix was carried out by solid-phase extraction under vaccuum. The purified drug was then collected directly into the vials of the HPLC autosampler. Chromatographic analysis was performed on a reversed-phase C18 column with water:acetonitrile (85:15 by vol) as the mobile phase and the UV detector set at 365 nm. The use of porfiromycin as internal standard provided a method with good within-day precision (CV 6.0% at 5 micrograms/L, n = 6), linearity (0.5-50 micrograms/L), and specificity. The lower limit of detection (< or = 0.5 microgram/L) proved to be suitable for plasma pharmacokinetics monitoring in two tested patients treated with MMC + HT for superficial bladder cancer.

Antibiotics, Antineoplastic↗
PubMed1996

Particulate naturally processed peptides prime a cytotoxic response against human melanoma in vitro.

For an efficient antitumor cytotoxic response, tumor antigenic peptides need to be presented by professional antigen-presenting cells in association with MHC class I molecules. We established in vitro short-term human CTL lines from healthy and melanoma-bearing subjects, using as antigen-presenting cells autologous adherent cells after phagocytosis of latex beads coated with melanoma peptides. Melanoma peptides were obtained by acid extraction of melanoma cells that matched with donor peripheral blood mononuclear cells, at least for one HLA-A allele. The cytotoxic activity of the lines was specific for the melanoma from which peptides were obtained and for melanoma sharing HLA alleles. These results demonstrate that a complex mixture of naturally processed melanoma peptides conjugated to a phagocytic substrate that targets them into the MHC class I pathway of adherent cells can prime a CTL response in healthy subjects in vitro, and that peptides from allogeneic tumors may be used to propagate CTL in melanoma patients. Our data support the feasibility of active and passive vaccination procedures with nonliving vaccines in cancer patients.

Animals↗
PubMed1996

Ceftazidime kinetics of diffusion in inoculated agar plates.

The ceftazidime concentration in agar plates inoculated with Pseudomonas aeruginosa was determined by high-performance liquid chromatography at fixed points (3, 6, 9, 12, and 15 mm) from the disk center and at fixed times (2, 4, 6, 16, and 24 h) to study the antibiotic kinetics of diffusion. A statistical difference between the concentrations determined in the presence of microorganisms and in uninoculated plates after 16 and 24 h was evidenced and was probably ascribable to the drug hydrolysis carried out by the induced beta-lactamase.

Agar↗
PubMed1995

Behavior of frozen serum pools and lyophilized sera in an external quality-assessment scheme.

To further improve analytical accuracy in clinical chemistry, proficiency testing needs amelioration in the quality of materials tested and in target value assignment. To obtain information on the actual state-of-the-art in the Lombardy region of Italy, and to examine the behavior of different types of control materials (fresh-frozen human sera and lyophilized materials), we developed the following experimental design. Two human serum pools and two lyophilized sera were distributed to 32 laboratories for determination of glucose, creatinine, cholesterol, sodium, potassium, and gamma-glutamyltransferase (gamma-GT). Each analyte was measured in triplicate on each of 3 days. Target values for the controls were obtained with Reference Methods. The results show a good intralaboratory precision for every component but some accuracy problems for glucose, creatinine, cholesterol, and gamma-GT. Lyophilized materials showed some commutability problems for glucose, electrolytes, and gamma-GT, mainly with dry chemistry technology.

Blood↗
PubMed1995

HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue.

This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N-ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (< 0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/- 4%), and linearity (r > 0.999). Samples, after PCA acidification, were stable at room temperature and 4 degrees C for 3 days, and at -20 degrees C and -80 degrees C for > 1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.

Animals↗
PubMed1994

Open-chain peptides obtained by acidic hydrolytic cleavage of cyclosporin A.

Hydrolysis of cyclosporin A (CsA) was studied in order to clarify the still undefined point of attack of the acidic degradation. Among ether extractable and water-soluble products formed from CsA in HCl, two open-chain peptides were isolated by high-performance liquid chromatography which were identified as the deca- and nonapeptides deriving from CsA through the hydrolytic cleavage of amino acid residue 11 and both residues 11 and 10, respectively. Identification was carried out by fast atom bombardment tandem mass spectrometry.

Amino Acid Sequence↗
PubMed1993

Capillary electrophoresis for protein analysis: separation of human growth hormone and human insulin molecular forms.

The capillary zone electrophoresis (CZE) technique was evaluated for separation and quantification of human growth hormone (hGH), human insulin (hI), and proinsulin. Three different molecular forms of biosynthetic hGH (20K, 22K, 44K), methionyl-hGH, biosynthetic hI, and proinsulin, were studied. The hormones were separated with uncoated capillaries, and various analytical conditions were tested (different buffers, ionic strength, pH). The samples were introduced both at positive pressure and electrokinetically, and the voltage applied was varied in each case. Linearity, repeatibility, and limit of detection (0.8 microM for hGH and 1.3 microM for insulin) were determined. Different purification steps were tried in order to find a suitable preanalytical procedure for hGH and hI purification from plasma. Recovery rates from 65 to 80% were obtained.

Electrophoresis↗
PubMed1992

High-performance liquid chromatographic method with fluorescence detection for the determination of total homocyst(e)ine in plasma.

A high-performance liquid chromatographic method for the determination of total plasma homocyst(e)ine [H(e)] after reduction with sodium tetrahydroborate and precolumn derivatization with o-phthaldialdehyde is described. The analyses, carried out on a reversed-phase C18 column, were based on spectrofluorimetric detection. The sensitivity was 1 pmol per injection and the intra- and inter-assay relative standard deviations were 1.8% and 5%, respectively. The plasma H(e) concentration determined in 40 healthy volunteers (20-60 years old) was 12.4 +/- 2.9 microM (mean +/- S.D.), in good agreement with reference values.

Adult↗
PubMed1992

Total urinary hydroxyproline determined with rapid and simple high-performance liquid chromatography.

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).

Aniline Compounds↗
PubMed1990

Determination of creatinine in serum and urine by a rapid liquid-chromatographic method.

We describe an HPLC ion-pair procedure for rapid and specific evaluation of creatinine in serum and urine. We used a 15 cm X 4.6 mm ODS column with a 50/50 (by vol) mixture of sodium decanesulfonic acid (10 mmol/L, pH 3.2) and methanol and measured absorbance at 236 nm. Serum (100 microL) or 30-fold-diluted urine (100 microL) was added to 400 microL of acetone. After centrifugation, the supernates (300 microL) were dried, reconstituted with the mobile phase, and injected into the HPLC. Assay precision was tested for concentrations of 10, 29, and 130 mg/L and yielded, respectively, 3.1%, 2.1%, and 1.1% for within-day CV and 2.8%, 2.1%, and 2.2% for total CV. Analytical recovery was 102 (+/- 6.7%). Linearity was demonstrated in the 0-200 mg/L range for serum and 0-3.5 g/L range for urine (r greater than or equal to 0.999). The detection limit for creatinine (signal-to-noise ratio = 3) was 0.5 mg/L. We used cimetidine for internal standardization. Correlation was good between this procedure and the Jaffé kinetic, the enzymatic (creatinine amidohydrolase), and the Fuller's earth alkaline picrate methods.

Chromatography, High Pressure Liquid↗
PubMed1989

Chromatofocusing and isoelectric focusing in immobilized pH gradients compared for characterization of human hemoglobin variants.

We compared the performance of two highly resolving methods, chromatofocusing (CRF) and isoelectric focusing in immobilized pH gradients (IPGF), for the separation of human hemoglobin variants. Lysates containing 13 different hemoglobins, including variants of clinical and geographical importance, and four electrophoretically "silent" variants (Hb Brockton, Hb Cheverly, Hb Köln, and Hb Waco) were analyzed. Both techniques showed a good intrarun precision (CV = 0.87% for CRF, 0.27% for IPGF) and high and similar resolving power (0.010 pH units, with the pH gradients used in this work). The use of an ultranarrow IPGF range (pH 7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution between Hb Brockton, Hb Köln, and Hb A. In some cases (Hb D-Los Angeles, Hb F, Hb Waco), the variants were separated from Hb A in different orders, depending on which technique was used, probably because of the different analytical principles of the two methods. As a second-level test, both procedures are informative for characterization of human hemoglobin variants.

Chromatography↗
PubMed1988

Pre-column derivatization of amino acids with N,N-diethyl-2,4-dinitro-5-fluoroaniline and reversed-phase liquid chromatographic separation.

A new method for the separation and quantification of primary and secondary amino acids by reversed-phase high-performance liquid chromatography and spectrophotometric detection is described. The use of the novel derivatizing reagent N,N-diethyl-2,4-dinitro-5-fluoroaniline yields derivatives that are more stable to light and heat in solution than the traditional ones. A simple gradient between acidic acetate buffer and acetonitrile allows the complete separation of 21 amino acids in 80 min at room temperature. The good reproducibility of peak areas and retention times allows the application of this simple and low-priced method to the determination of amino acids in the range 50-500 pmol per residue.

Amino Acids↗
PubMedDate not supplied

Development and characterization of pituitary GH3 cell clones stably transfected with a human proinsulin cDNA.

Successful beta-cell replacement therapy in insulin-dependent (type I) diabetes is hindered by the scarcity of human donor tissue and by the recurrence of autoimmune destruction of transplanted beta cells. Availability of non-beta cells, capable of releasing insulin and escaping autoimmune recognition, would therefore be important for diabetes cell therapy. We developed rat pituitary GH3 cells stably transfected with a furin-cleavable human proinsulin cDNA linked to the rat PRL promoter. Two clones (InsGH3/clone 1 and 7) were characterized in vitro with regard to basal and stimulated insulin release and proinsulin transgene expression. Mature insulin secretion was obtained in both clones, accounting for about 40% of total released (pro)insulin-like products. Immunocytochemistry of InsGH3 cells showed a cytoplasmic granular insulin staining that colocalized with secretogranin II (SGII) immunoreactivity. InsGH3 cells/clone 7 contained and released in vitro significantly more insulin than clone 1. Secretagogue-stimulated insulin secretion was observed in both InsGH3 clones either under static or dynamic conditions, indicating that insulin was targeted also to the regulated secretory pathway. Proinsulin mRNA levels were elevated in InsGH3 cells, being significantly higher than in betaTC3 cells. Moreover, proinsulin gene expression increased in response to various stimuli, thereby showing the regulation of the transfected gene at the transcriptional level. In conclusion, these data point to InsGH3 cells as a potential beta-cell surrogate even though additional engineering is required to instruct them to release insulin in response to physiologic stimulations.

Animals↗