A cell-based approach for the early assessment of the phospholipidogenic potential in pharmaceutical research and drug development.

Phospholipidosis is a term commonly used to indicate a phospholipid storage disorder; in affected cells, phospholipids accumulate in lysosomes that acquire a multilamellar morphological appearance. Cationic amphiphilic drugs (CADs) are suggested to induce phospholipidosis by direct interaction of xenobiotics with intracellular phospholipids or by the action of xenobiotics on the synthesis and metabolism of phospholipids. To date, electron microscopy (EM) represents the most reliable and the preferred method for the demonstration of phospholipidotic cell damage. Nevertheless, EM has a low throughput, it is expensive, and it is not suitable for screening purposes. We discuss here the assessment of the the phospholipidogenic potential of drugs using a cell culture-based model. In this test, intracellular phospholipids of treated U-937 cells (a human monocyte-derived cell line) were measured using the fluorescent probe Nile red. Eleven CADs reported to induce phospholipidosis in vivo and eight nonphospholipidogenic drugs were tested. Results obtained with the U-937 model confirmed the phospholipidogenic potential of drugs tested as described in the literature. Results have also been correlated with data obtained with a physical-chemical model (chromatographic hydrophobicity index measurement). Good correlation was obtained, confirming that the physical-chemical properties of CADs play a crucial role in the development of phospholipidosis. This work demonstrates that the U-937 model is a rapid and sensitive method for the determination of phospholipidosis-mediated cell damage. The specificity and the predictive potency observed make this method suitable for screening purposes in pharmaceutical development.

Field-amplified sample stacking capillary zone electrophoresis applied to the analysis of opiate drugs in hair.

The present paper describes the methodological optimization and validation of capillary zone electrophoresis (CZE) for the determination of major opiates (morphine, codeine, 6-monoacetylmorphine, acetylcodeine, heroin) in hair samples by using a field-amplified sample stacking injection before the separation in a binary running buffer (0.1 M sodium phosphate, pH 2.5, with 40% ethylene glycol). The applied potential was 20 kV, at 25 degrees C. Detection was by UV absorption at the fixed wavelength of 214 nm or by recording the full spectrum between 190-400 nm, thus improving the analytical selectivity and identification power of CZE. Hair samples were liquid/liquid extracted; dried extracts, reconstituted with a low-conductivity solvent (0.1 mM phosphoric acid, with 80% 1-propanol), were injected by electromigration at 10 kV for 99 s, after a 0.5 mm plug of water. Under the described conditions, the limit of detection (with a signal-to-noise ratio of 3) in hair extracts was 100 pg/mL for codeine, 75 pg/mL for morphine and 6-monoacetylmorphine (6-MAM), 150 pg/mL for ethylmorphine, and 0.75 ng/mL for acetylcodeine and heroin. The precision of the method was validated for standards in pure solution by using internal standardization, providing for intraday and day-to-day assays, in terms of migration times, relative standard deviation (RSD) values < or = 0.2%, and in terms of peak areas, RSD values <5.71%.

Nutritional supplementation with copper in the rat. I. Effects on adjuvant arthritis development and on some in vivo- and ex vivo-markers of blood neutrophils.

OBJECTIVE AND DESIGN: The aims of the work were: 1) to confirm the preliminarily observed anti-arthritic potential of a 200 ppm copper-supplemented diet in the rat: 2) to study the impact of the nutritional treatment and of the experimental pathology on neutrophil activity. ANIMALS AND CELLS: Two hundred female Sprague-Dawley rats were used. Polymorphonuclear leukocytes were isolated from these animals for the ex vivo studies. TREATMENT: Control-rats were maintained on a standard diet containing 5 ppm of copper. Supplemented-rats were kept on a diet containing 200 ppm of the metal. METHODS: Mycobacterium butyricum-induced arthritis was studied. Flame atomic absorption spectroscopy was used to assess copper and zinc levels. The "microplate-assay" technique was used to determine serum lysozyme concentration (lysis of Micrococcus lysodeikticus cell walls), as well as neutrophil O2- generation (superoxide dismutase-inhibitable reduction of ferricytochrome-c), and adhesion (activity of the membrane enzyme acid phosphatase). The results were statistically evaluated by the Student's t test. RESULTS: The nutritional copper-supplementation: 1) significantly inhibited the adjuvant-arthritis development (33% +/- 5, P<0.01); 2) did not modify lysozyme secretion or superoxide production; 3) significantly decreased the percentage of cell adhesion by an average of 41% +/- 19 (P<0.01). CONCLUSIONS: The copper-supplemented diet has an anti-arthritic effect which may be also primed by the effect of copper on the expression of the neutrophil cell-adhesion molecules.

Influence of an acute exercise on neutrophil and platelet adhesion, nitric oxide plasma metabolites in inactive and active subjects.

In this work we studied the influence of an acute exercise either on nitrite/nitrate plasma levels or on neutrophil and platelet adhesion in inactive and active subjects. Twelve healthy subjects (6 inactives and 6 actives) exercised on a racing cycle ergometer performing stepwise increases in intensity until reaching, within 5 min, a heart rate of 150 beats x min(-1) which represents an oxygen consumption of about 75 % of the individual maximum rate of oxygen uptake. From peripheral venous blood samples (drawn from all subjects before, immediately after the end of exercise, and 1 hour later) neutrophils and platelets were isolated to test plate adhesion, and nitrite/nitrate concentrations were measured in the plasma. Immediately after the acute exercise, in active subjects we observed a significant decrease in the percentage of neutrophil adhesion (7.96+/-2.38 vs. 14.10+/-3.14), associated with an increase in nitrite/nitrate plasma levels (81.38+/-10.76 vs. 41.08+/-8.13 micromol x l(-1)), restored by a 40 min pre-incubation with NG-nitro-L-arginine methyl ester (L-NAME). In unstimulated platelets we observed a significant lower percentage of platelet adhesion in active subjects compared to inactives after exercise. With thrombin or adenosine 5'-diphosphate as agonists platelet adhesion did not result significantly different in active subjects compared to inactives. In conclusion, our data show that physical exercise can induce changes in some cell activities, even if transient, and favour the generation of nitric oxide. The lower adhesion of neutrophils and platelets induced by regular exercise could be an important goal in the prevention of vascular and inflammatory diseases.

Improved method for carbohydrate-deficient transferrin determination in human serum by capillary zone electrophoresis.

Carbohydrate-deficient transferrin (CDT) is a reliable marker of chronic or repeated alcohol abuse. It indicates a group of isoforms of human transferrin (Tf), the main iron transport serum protein, deficient in sialic acid residues (asialo-, monosialo- and disialo-Tf) in comparison to the main isotransferrin which contains four sialic acid groups (tetrasialo-Tf). The aim of the present work was to develop a capillary electrophoretic method suitable for rapid determination of CDT components in serum. Serum samples (0.1 ml) were saturated with iron by incubation with 10 mM FeCl3 (2 microl) and 500 mM NaHCO3 (3 microl) for 30 min, then diluted 1:10 in water and injected by positive pressure (0.5 p.s.i. for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (57 cm x 20 microm I.D.) and a buffer composed of 100 mM sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 mM diaminobutane. Applied voltage was 20 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were baseline separated. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-Tf, and 0.5% of trisialo-Tf, expressed as percentages of the terasialo-Tf peak area. Day-to-day RSDs of relative migration times were < or = 0.2%. Quantitation showed day-to-day RDSs < or = 6.9% and < or = 10.9% for disialo- and trisialo-Tf, respectively. The results from 79 control subjects, including social drinkers, and 23 alcoholics showed disialo- and trisialo-Tf significantly increased in patients (P<0.0001 and <0.01, respectively). A clear interference from trisialo-Tf in an immunoassay for CDT was demonstrated. The present method is suitable for confirmation of CDT immunoassays by independent technique.

Capillary electrophoresis: a new analytical tool for forensic toxicologists.

In capillary electrophoresis, electrophoretic or electrokinetic separations are carried out in tiny capillaries at high voltages (10-30 kV), thus achieving high efficiency (N > 105), resolution power, and mass sensitivity (down to 10(-18)-10(-20) moles). The main characteristics of capillary electrophoresis are versatility of application (from inorganic ions to large DNA fragments), use of different separation modes with different selectivity, low demands on sample volume, negligible running costs, possibility of interfacing with different detection systems including mass spectrometry, and the ruggedness and simplicity of the instrumentation. Capillary electrophoresis applications in the forensic sciences are now rapidly growing, particularly in forensic toxicology. The present paper briefly describes the basic principles of capillary electrophoresis and presents a selected review of its main applications to the analysis of illicit/controlled drugs in biologic samples. An original analytical approach to the determination of carbohydrate deficient transferrin, a new marker of chronic alcohol abuse, based on capillary electrophoresis is also described. It is concluded that the peculiar separation mechanisms and the high complementarity of capillary electrophoresis to chromatography make it a new powerful tool of investigation in the hands of forensic toxicologists.

Hair analysis by using radioimmunoassay, high-performance liquid chromatography and capillary electrophoresis to investigate chronic exposure to heroin, cocaine and/or ecstasy in applicants for driving licences.

The present paper describes an integrated diagnostic strategy to check the physical fitness of subjects, formerly users of illicit drugs, to obtain a driving license, after having quit their addiction. According to the Italian law, applicants for a driving license with a history of drug abuse must give evidence to have quit this behaviour and to show no risk of relapse in the future. To prove this, at our institute, they undergo medical examination, hair analysis and a urinalysis program on eight seriate samples, collected over about 40 days. About 700 subjects per year are investigated with this strategy. The hair samples are screened for opiates (morphine), cocaine and ecstasy, the most abused illicit substances in our region, by using commercial radioimmunoassays adopting cut-off levels of 0.1 ng/mg. All positive samples and about 10% of negatives are confirmed by high-performance liquid chromatography. Further confirmation of results can be carried out by capillary electrophoresis (and/or GC/MS or MS/MS). In 1998, the prevalence of positives for morphine, cocaine and ecstasy was 4.8, 11.3 and 2.6%, respectively. In this year, for the first time, the percentage of hair samples positive for cocaine was greater than that for opiates. The results of this integrated diagnostic strategy are presented and discussed, with particular emphasis on the comparison between hair analysis on a single sample and seriate urinalyses (on eight samples).

Objective Diagnosis of Chronic Alcohol Abuse - Determination of Carbohydrate-Deficient Transferrin (CDT) with Capillary Electrophoresis.

To assist the diagnosis of high risk alcohol consumption or alcohol dependence, particularly in the absence of evidences of recent alcohol intake (relapse), objective biochemical markers are today available, which, if used correctly, may reduce the degree of subjectivity that the adoption of merely clinical and psychological diagnostic criteria shows inevitably. The present paper reviews briefly in terms of diagnostic sensitivity and specificity the most important conventional markers of chronic alcohol abuse - e.g., γ-glutamyl transferase (GGT), erythrocyte mean corpuscular volume (MCV), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) - as well as those more recently proposed, namely aldehyde dehydrogenase (ALDH), high density lipoprotein cholesterol, serum triglycerides, urate, fatty acid ethyl and methyl esters, phosphatidylethanol, dolichols, ß-hexosaminidase and protein acetaldehyde adducts. However, the specific focus of this review is on carbohydratedeficient transferrin (CDT), the collective name of a group of transferrin isoforms lacking totally or partly the oligosaccharide chains usually linked at two glycosylation sites in the C domain of the protein. CDT, at present, is considered the most reliable marker of excessive alcohol intake for at least a week, and has a chronological diagnostic window of about two weeks before sample collection. CDT is reviewed in terms of diagnostic value, applications and, particularly, in the different analytical approaches, with special emphasis on capillary electrophoresis, the latest method proposed for its quantitative determination.

Increased endogenous nitric oxide production induced by physical exercise in peripheral arterial occlusive disease patients.

The effects of 14-day physical exercise or iloprost treatment (0.5-2 ng/Kg/min) on endogenous nitric oxide production and neutrophil adhesion were evaluated in 20 patients with peripheral arterial occlusive disease (Fontaine Stage II). Peripheral venous blood samples and 4-h urine samples were collected before, immediately after 14 days of therapy and 7-10 days after therapy in order to evaluate neutrophil adhesion, nitrite/nitrate and cGMP excretion rates. A longer pain free walking distance was observed after exercise, compared to iloprost (>500 m in 3/10 subjects). Urinary nitrite/nitrate, as well as cGMP concentrations, significantly increased after exercise. Nitrite/nitrate excretion rate inversely correlated to neutrophil adhesion. No variations were observed in these parameters in iloprost treated patients. The improvement in claudication and the transient increase in urinary nitrite/nitrate suggest a possible nitric oxide-dependent mechanism for the clinical efficacy of physical exercise. The results from the present and previous observations indicate that, besides pharmacological treatments, a regular aerobic exercise improves peripheral arterial occlusive disease.

Carbohydrate-deficient transferrin determination revisited with capillary electrophoresis: a new biochemical marker of chronic alcohol abuse.

To aid in the objective diagnosis of chronic alcohol abuse in both clinical and medico-legal environments, reliable biochemical markers are needed. In addition to the conventional indicators, which include y-glutamyl transferase, erythrocyte mean corpuscular volume, aspartate aminotransferase, and alanine aminotransferase, carbohydrate-deficient transferrin (CDT) has recently been introduced. According to a reliable body of literature, CDT is the most reliable marker of chronic excessive alcohol intake. CDT is the collective name for minor isoforms of human transferrin, and therefore highly selective and sensitive methods are required for its analysis in serum. Moreover, the need for precise quantification poses additional problems of accuracy and precision. For these purposes, capillary electrophoresis shows excellent potential in terms of quantitative accuracy, precision, speed, automation, economy, and simplicity of operation. The simple, optimized capillary zone electrophoretic analysis of CDT in human serum is discussed in this paper and is compared to the traditional analytical method based on microcolumn ion exchange chromatography followed by immunoassay.

Biotransformation and cytotoxic properties of NO-donors on MCF7 and U251 cell lines.

Previous studies have shown a role for nitric oxide (NO) as a cytotoxic effector. In the present work, two chemically different NO-donors such as glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP) were evaluated for both NO release and cytostatic/cytotoxic properties. Nitrite accumulation in the supernatant of MCF-7 and U251 cell lines indicated a greater and quickly release of NO derived from SNAP. A time-course of hemoglobin absorption spectral changes showed a greater release of NO derived from GTN in presence of cells compared to the values observed in the media, confirming that the release of NO by GTN can be enzymatic and non-enzymatic. On the contrary, SNAP generated NO without contribution of cellular components and saturated oxyhemoglobin quickly, within 2 hours. Both NO-donors inhibited thymidine incorporation in a similar manner and dose-dependently in U251 cells, but not in MCF-7 cells, where SNAP at the highest tested dose of 1000 microM induced only a 33% cytostatic effect. About trypan blue exclusion test, after 24 h GTN and SNAP, releasing similar amounts of NO, showed comparable cytotoxic effects on U251 cells (50% dead cells), but not on MCF-7 cells, where GTN resulted more cytotoxic. From our data, the "in vitro" antitumoral activity of NO-donors seems to be related to the type of tumor cell lines, to the amount and duration of NO release.

Optimized determination of carbohydrate-deficient transferrin isoforms in serum by capillary zone electrophoresis.

Carbohydrate deficient transferrin (CDT) is one of the most reliable markers of chronic alcohol abuse. It consists of a group of minor isoforms of human transferrin (the main iron transport serum protein) deficient in sialic acid groups (asialo, monosialo and disialo) with a pI > 5.7, while the main isotransferrin (tetrasialo) has a pI of 5.4. The aim of the present work was to develop a capillary electrophoretic method to determine CDT in serum, suitable for routine use as a confirmatory technique of the current screening methods based on immunoassays. Serum samples (0.5 mL) were saturated with iron by incubation with 10 mM FeCl3 (9 microL) and 500 mM NaHCO3 (12 microL) for 30 min, then diluted 1/10 in water and injected by positive pressure (0.5 psi for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (20 microm ID, 37 cm in length) and a buffer composed of 100 mM sodium tetraborate adjusted with boric acid to pH 8.3. Applied voltage was 10 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were separated in human serum. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-transferrin, and 0.4% of trisialo-transferrin, expressed as percentages of the terasialo-transferrin peak area. Relative standard deviations (RSD) of absolute migration times were < 1%, while RSD of relative migration times (on the basis of tetrasialo-transferrin) were < 0.1%. Intra-day and day-to-day peak quantitation precision studies showed RDS ranging from 4 to 9% and from 13 to 24% for disialo- and trisialo-transferrin, respectively. The results from 30 control subjects, including social drinkers, and 13 alcoholics showed disialo- and trisialo-transferrin significantly increased in patients by a factor of about 4.5 (P < 0.0001).

Hair analysis for abused drugs by capillary zone electrophoresis with field-amplified sample stacking.

The present paper describes the methodological optimisation and validation of a capillary zone electrophoresis method for the determination of morphine, cocaine and 3,4-methylenedioxymethamphetamine (MDMA) in hair, with injection based on field-amplified sample stacking. Diode array UV absorption detection was used to improve analytical selectivity and identification power. Analytical conditions: running buffer 100 mM potassium phosphate adjusted to pH 2.5 with phosphoric acid, applied potential 10 kV, temperature 20 degrees C, injection by electromigration at 10 kV for 10 s, detection by UV absorption at the fixed wavelength of 200 nm or by recording the full spectrum between 190 and 400 nm. Injection conditions: the dried hair extracts were reconstituted with a low-conductivity solvent (0.1 mM formic acid), the injection end of the capillary was dipped in water for 5 s without applying pressure (external rinse step), then a plug of 0.1 mM phosphoric acid was loaded by applying 0.5 psi for 10 s and, finally, the sample was injected electrokinetically at 10 kV for 10 s. Under the described conditions, the limit of detection was 2 ng/ml for MDMA, 8 ng/ml for cocaine and 6 ng/ml for morphine (with a signal-to-noise ratio of 5). The lowest concentration suitable for recording interpretable spectra was about 10-20-times the limit of detection of each analyte. The intraday and day-to-day reproducibility of migration times (n = 6), with internal standardisation, was characterised by R.S.D. values < or = 0.6%; peak area R.S.D.s were better than 10% in intraday and than 15% in day-to-day experiments. Analytical linearity was good with R2 better than 0.9990 for all the analytes.

Neutrophil behavior following exposure to in vivo or in vitro zinc in normal and acutely-inflamed rats: studies on lysozyme secretion, superoxide anion release and platelet adhesion.

The mechanism was studied of the anti-inflammatory effect of oral zinc (114 mg/kg/day of elemental metal, given for 14 days) on the development of the carrageenan-induced paw oedema of the rat, and the impact of in vivo treatment on the activity of neutrophils isolated from the blood of inflamed and non-inflamed animals. The effects of the in vitro incubation with the metal on either non-inflamed or inflamed neutrophils coming from zinc-untreated rats were also examined. It was found that the administration of oral zinc inhibited markedly the process of ex vivo adhesion of the cells obtained from the inflamed rats (an observation confirmed by the in vitro experiments). In vitro release of lysozyme and superoxide anion productions were measured: in the absence of zinc, the 30' of pre-incubation carried out before stimulating with PMA did not influence the cell's reactivity of the non-inflamed neutrophils. It was, on the contrary, capable of significantly reducing that of the inflamed ones. As a consequence, it is quite difficult to properly interpret the data obtained studying the activity of the cells exposed to the metal in vitro.

Role of endogenous and exogenous nitric oxide on intestinal mucosa and microflora in the rat.

In the present study the effects of exogenous and endogenous nitric oxide (NO) on intestinal bacteria and on the intestinal tissue integrity have been investigated in healthy rats and in rats receiving bacterial endotoxin (LPS). A segment of jejunum was taken in order to evaluate tissue damage and hematoxylin-eosin staining; microbiological studies were carried out collecting stool samples. Administration of LPS (5 mg kg-1 i.v.) induced a moderate jejunal damage, which was completely prevented by NG-nitro-L-arginine methyl ester (L-NAME), 5 mg kg-1 s.c.), thus suggesting a damage of endogenous NO on the intestinal mucosa; sodium nitroprusside (SNP, 10 mg kg-1 os) reduced significantly jejunal damage induced by LPS. Endogenous NO produced by the administration of LPS resulted to be cytotoxic for all examined aerobic and anaerobic bacteria, while exogenous NO, released from SNP, showed an inhibitory effect only on Entero. faecalis and E. coli growth. From our data, it seems reasonable to conclude that high local levels of NO are required in order to observe jejunal damage and cytotoxic effects on aerobic and anaerobic faecal flora.

Pharmacokinetics of a new nitroderivative of acetylsalicylic acid after a single dose in rats.

The pharmacokinetics of a new ASA-nitroderivative compound, NCX 4016 (ASA-NO2), was evaluated using an HPLC method. After single equimolar doses of ASA (35 mg kg(-1)) or ASA-NO2 (65 mg kg(-1)), no detectable levels of these compounds have been observed in rat plasma samples. SA peak levels were obtained at 3 h and 6 h after ASA and ASA-NO2 administration respectively. The elimination rate constants of SA were similar after ASA and ASA-NO2, suggesting a similar elimination phase of this metabolite in rats. From these data it is evident that ASA-NO2 is slowly metabolized in ASA, which is rapidly converted to SA.

Vasodilating properties of a new non-steroidal anti-inflammatory drug, nitroflurbiprofen, on rat aortic rings.

The effects of nitroflurbiprofen (NFP), a new non-steroidal anti-inflammatory drug containing a nitroxybutyl moiety, on rat aortic rings were compared with those of flurbiprofen (FP) and glyceryl trinitrate (GTN). NFP and GTN relaxed, in a dose-dependent manner, either intact or rubbed aortic rings precontracted with epinephrine. Pretreatment with FP did not influence the relaxant activity of NFP in both endothelium-intact and -denuded arteries. In unrubbed preparations, FP did not affect contraction induced by epinephrine, while in rubbed ones a moderate relaxation was observed. Methylene blue and oxyhaemoglobin completely reversed the vasodilating effect of NFP in both rubbed and unrubbed preparations. Moreover, the addition of cysteine 5 mmol l-1 at the end of the cumulative application of NFP resulted in a further relaxation of aortic rings. These results indicate that NFP possesses vasodilatory activity which appears to be dependent on the release of nitric oxide or nitric oxide derivatives.

Effect of single and repeated doses of a new nitroderivative of acetylsalicylic acid on platelet TXA2 production in rats.

The effects of a new ASA-nitroderivative compound, NCX 4016 (ASA-NO2), on platelet TXA2 synthesis after single and repeated doses in the rat were investigated. Compared to ASA, cumulative doses of ASA-NO2 showed similar inhibitory effects on platelet TXA2 synthesis and significant increases in nitrite/nitrate plasma concentrations 1 h after the last drug administration: 24 h later nitrite/nitrate plasma levels returned to the control values, while serum TXA2 concentrations did not change. A time-course study after a single dose of ASA-NO2 showed a significant inhibition of platelet TXA2 production also 24 h after drug administration and a significant increase in nitrite/nitrate plasma levels until 10 h.