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Biomedical subjects

M Montagna

Publications and source records attributed to M Montagna.

At least 19 recordsLinked to original sources
PubMed2002

Hair analysis for opiates, cocaine and metabolites. Evaluation of a method by interlaboratory comparison.

The aim of this study was to evaluate the performance of a technique for the simultaneous testing of opiates, cocaine and metabolites in hair by interlaboratory comparison. Sixteen forensic and clinical laboratories with different degrees of experience in hair analysis participated voluntarily in the study (no selection criteria were applied). The suggested analytical procedure, the one routinely used in our laboratory, consisted of incubation in HCl 0.1N (45 degrees C, overnight), solid phase extraction (with Bond Elut Certify) cartridges), derivatisation (trimethylsilyl (TMS) derivatives) and GC-MS analysis. Three different mixtures of finely cut (1 mm or less) hair were prepared using drug-users' and drug-free hair: one 'negative' sample (<0.1 ng/mg for morphine, 6-acetylmorphine (6AM), cocaine and benzoylecgonine (BE)), one 'low concentration' sample (between 0.5 and 2 ng/mg) and one 'high concentration' sample (>3 ng/mg). Accuracy and precision (CV% lower than 5.1, 9.9, 5.2, 3.8, 7.3 and 8.3% for morphine, 6AM, codeine, cocaine, BE, and methylecgonine (ME), respectively; range 0.5-5 ng/mg) of the method and homogeneity of the mixtures were evaluated in our laboratory by intraday (CV% lower than 12% for all analytes) and interday analyses (CV% lower than 17% for all analytes except 6AM, 25%). Participants in the study were grouped into: (1) laboratories (n = 6) obtaining the best qualitative and quantitative values, corresponding to those with long experience in hair analysis; (2) laboratories (n = 5) with no reported false positive and/or false negatives; (3) laboratories (n = 5) with one or more reported false positives/false negatives. The results obtained by the labs of the first group were used as reference values. The scatter of data was similar to those obtained in other published studies.

Cocaine↗
PubMed2001

Lack of association between androgen receptor CAG polymorphism and familial breast/ovarian cancer.

The human androgen receptor (AR) gene contains a highly polymorphic CAG repeat in exon 1 that is inversely correlated with AR transcriptional activity in vitro. Several studies have shown that fewer CAG repeats are associated with an increased risk as well as more aggressive forms of prostate cancer. More recently, AR allele length was also inversely correlated with the histological grade of breast cancer, but no association was found between the AR-CAG polymorphism and the risk of either breast or ovary cancer. On the contrary, it was proposed that a longer CAG repeat sequence might be associated with an increased risk of breast cancer in BRCA1 mutation carriers, thus suggesting a different role of the AR-CAG polymorphism in sporadic and inherited breast cancers. With the intent of better understanding the role of the AR-CAG polymorphism as a cancer risk modifier, we defined the AR genotype of 151 patients (101 with breast and 50 with ovary cancer) belonging to high-risk breast/ovary cancer families. No difference in CAG repeat length was found between either breast and ovary cancer patients or age at diagnosis of both tumors. These results were also confirmed in a sub-group of 47 breast cancer cases, that either carried a BRCA gene mutation (11 cases) or were identified by very stringent operational criteria as hereditary breast cancers. Even though a substantially larger sample size would be required to reach conclusive evidence, our findings suggest that the AR-CAG polymorphism does not act as a modifier of tumor onset or tumor phenotype in breast/ovarian cancer families.

Alleles↗
PubMed2001

Color bar coding the BRCA1 gene on combed DNA: a useful strategy for detecting large gene rearrangements.

Genetic linkage data have shown that alterations of the BRCA1 gene are responsible for the majority of hereditary breast and ovarian cancers. BRCA1 germline mutations, however, are found less frequently than expected. Mutation detection strategies, which are generally based on the polymerase chain reaction, therefore focus on point and small gene alterations. These approaches do not allow for the detection of large gene rearrangements, which also can be involved in BRCA1 alterations. Indeed, a few of them, spread over the entire BRCA1 gene, have been detected recently by Southern blotting or transcript analysis. We have developed an alternative strategy allowing a panoramic view of the BRCA1 gene, based on dynamic molecular combing and the design of a full four-color bar code of the BRCA1 region. The strategy was tested with the study of four large BRCA1 rearrangements previously reported. In addition, when screening a series of 10 breast and ovarian cancer families negatively tested for point mutation in BRCA1/2, we found an unreported 17-kb BRCA1 duplication encompassing exons 3 to 8. The detection of rearrangements as small as 2 to 6 kb with respect to the normal size of the studied fragment is achieved when the BRCA1 region is divided into 10 fragments. In addition, as the BRCA1 bar code is a morphologic approach, the direct observation of complex and likely underreported rearrangements, such as inversions and insertions, becomes possible.

Breast Neoplasms↗
PubMed2001

Comprehensive drug screening by integrated use of gas chromatography/mass spectrometry and Remedi HS.

The authors evaluated an integrated approach for the screening of drugs in biosamples consisting of gas chromatography/mass spectrometry analysis of serum or whole blood (SB/GC-MS) and of high-performance liquid chromatographic and ultraviolet (HPLC-UV) analysis of urine with the REMEDi HS Biorad system (U/REM) (Bio-rad; Segrate, MI, Italy). Urine and blood samples from 26 suspected intoxicated patients and from 22 suspected lethal poisoning cases were examined. Eighty-one of the 99 parent drugs/main metabolites detected were identified by SB/GC-MS and 54 with U/REM. Thirty-six drugs/metabolites were identified with both methods, 45 by SB/GC-MS alone, and 18 by U/REM alone. Absence of the mass spectrometry (MS) spectra in the reference library and high polarity of the analytes were the main reasons for failed identification by SB/GC-MS. Unsuccessful identifications with U/REM were basically caused by the absence of the UV spectra in the reference library or by low chromatographic and spectroscopic selectivity as in the case of barbiturates and benzodiazepines (BZD), which represented 11% and 51%, respectively, of the 45 SB/GC-MS unique identifications. Urine samples of 14 BZD-positive cases were also submitted to enzymatic hydrolysis and analyzed with the REMEDi UBz assay, and results were compared with those obtained by SB/GC-MS: 14 of the 22 identified BZD were detected with both methods, three by U/REM only, and five by SB/GC-MS only. In conclusion, the integrated use of SB/GC-MS and U/REM approaches greatly enhances the amount and quality of analytical information obtainable by applying either method alone.

Chromatography, High Pressure Liquid↗
PubMed2001

Antiretrovirals: simultaneous determination of five protease inhibitors and three nonnucleoside transcriptase inhibitors in human plasma by a rapid high-performance liquid chromatography--mass spectrometry assay.

An analytical technique using liquid chromatography (LC) coupled with electrospray-mass spectrometry (ESI--MS) has been developed for the simultaneous determination of five protease inhibitors (PIs): saquinavir, indinavir, ritonavir, nelfinavir, and amprenavir; and three non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine, delavirdine, and efavirenz, in human plasma. This assay allows the elution and identification of these drugs in a single run (10 minutes) using a linear gradient with water and acetonitrile. The procedure involves liquid--liquid extraction. High-performance liquid chromatography (HPLC) separation was achieved on a C18 reversed-phase column, with a linear gradient elution followed by mass spectrometry detection. The calibration curves, obtained by automatic process peak area integration, show a good linearity in a range of concentrations between 20 and 10,000 ng/mL (40--10,000 ng/mL for efavirenz). The limit of detection was approximately 10 ng/mL for seven drugs (25 ng/mL for efavirenz). The coefficients of variation (CV) were always less than 15% for both intraday and interday precision for each compound. The recovery of the eight drugs ranged from 88.5% to 100%. This novel LC/ESI--MS assay provides an excellent method for simultaneous quantitative monitoring of different components of the highly active antiretroviral treatments (HAARTs) in patients treated simultaneously with PIs and NNRTIs, and it has been successfully applied to therapeutic drug monitoring and pharmacokinetic studies.

Alkynes↗
PubMed2001

Nelfinavir suspension obtained from nelfinavir tablets has equivalent pharmacokinetic profile.

The pharmacokinetics of nelfinavir tablets (A) and an oral simplified nelfinavir suspension (B) were studied. Twelve healthy volunteers randomly received either five 250-mg nelfinavir tablets or a simplified oral suspension obtained from tablets dissolved in water (nelfinavir 1250 mg in 100 mL of water) in a single dose before being crossed over to the second treatment after a one-week washout period. Blood samples were drawn up to 24 h after drug administration. Nelfinavir concentrations in plasma were analyzed by a specific and validated reverse-phase high-performance liquid chromatography assay (HPLC) with UV detection, and pharmacokinetic values were determined. For the AUC(0-infinity) with means+/-SD of 31.71+/-7.85, 30.88+/-10.28 (microg/L) respectively for treatments B and A, the ratio (F(B/A)) was of 1.1 with a C.I. of 0.90-1.24. For Cmax with means+/-SD of 3.1+/-0.6 (treatment B) and 3.2+/-0.8 mg/mL (treatment A), the ratio was 1.0. with C.I. of 0.92-1.08. The two treatments evidenced no significant differences in AUC(0-inifnity) and Cmax values and the two-one sided t-test showed that the two preparations are bioequivalent. There was no significant difference in Tmax between the liquid and tablets. Nelfinavir suspension might be a option for treating HIV-infected patients with swallowing disturbances or compliance problems.

Administration, Oral↗
PubMed2001

Comparison of the plasma pharmacokinetics of lamivudine during twice and once daily administration in patients with HIV.

OBJECTIVE: To compare the plasma pharmacokinetics of lamivudine 150mg twice daily and 300mg once daily in patients with HIV-1 infection. DESIGN: Nonblind, sequential, pharmacokinetic study. PARTICIPANTS: 13 patients with HIV-1 infection (median age 36 years). METHODS: Patients were tested during twice daily and then once daily regimens of lamivudine. In both regimens, the total daily dose of lamivudine was identical (300 mg/day). Blood samples for pharmacokinetic analysis were taken over a 12-hour period after > or =7 days of twice daily administration, and again over a 24-hour period after 7 days of once daily administration,. RESULTS: 12 patients completed the study. Lamivudine pharmacokinetic parameters (mean +/- SD) after administration of 150mg twice daily were: peak plasma concentration (Cmax) 2077+/-816 microg/L; trough plasma concentration (Cmin) 332+/-219 microg/L; elimination half-life (t 1/2beta) 6.1+/-1.9h; time to Cmax (t(max)) 1.6+/-0.7h; average concentration over the dosage interval (Cav) 711+/-269 microg/L; and area under the concentration-time curve (AUC) over 2 dosage intervals (24h) 17085+/-6464 microg x h/L. Corresponding values after administration of 300mg once daily were: Cmax 3461+/-854 microg/L; Cmin 146+/-87 microg/L; t1/2 7.9+/-3.4h; t(max) 2.2+/-1.3h; Cav 705+/-177 microg/L; and AUC over 1 dosage interval (24h) 16644+/-4150 microg x h/L. Statistical analysis showed a significant difference (p < 0.05) between the 2 schedules for Cmax and Cmin values, whereas no significant differences emerged for the other parameters. CONCLUSIONS: Once daily lamivudine leads to a similar exposure in plasma as twice daily administration of the same total daily dose. Since once daily administration may result in improved compliance, these results provide the pharmacokinetic basis for using lamivudine in a once daily regimen. Randomised clinical studies are needed to confirm this pharmacokinetic finding.

Adult↗
PubMed2000

Nature of the short wavelength excitations in vitreous silica: An X-Ray brillouin scattering study

The dynamical structure factor [ S(Q,E)] of vitreous silica has been measured by inelastic x-ray scattering varying the exchanged wave vector ( Q) at fixed exchanged energy ( E)-an experimental procedure that, contrary to the usual one at constant Q, provides spectra with much better identified inelastic features. This allows us to obtain the first direct evidence of Brillouin peaks in the S(Q,E) of SiO2 at energies above the boson peak (BP) energy, a finding that excludes the possibility that the BP marks the transition from propagating to localized dynamics in glasses.

Journal Article↗
PubMed2000

Frustration and sound attenuation in structural glasses

Three classes of harmonic disorder systems (Lennard-Jones-like glasses, percolators above threshold, and spring disordered lattices) have been numerically investigated in order to clarify the effect of different types of disorder on the mechanism of high frequency sound attenuation. We introduce the concept of frustration in structural glasses as a measure of the internal stress, and find a strong correlation between the degree of frustration and the exponent alpha that characterizes the momentum dependence of the sound attenuation gamma(Q) approximately Qalpha. In particular, alpha decreases from approximately d+1 in low-frustration systems (where d is the spectral dimension) to approximately 2 for high-frustration systems such as the realistic glasses examined.

Journal Article↗
PubMed2000

Simultaneous hair testing for opiates, cocaine, and metabolites by GC-MS: a survey of applicants for driving licenses with a history of drug use.

A sensitive GC-MS method for the simultaneous determination of opiates, cocaine, and metabolites in hair at a cut-off level of 0.1 ng/mg was adopted to assess past exposure to these drugs in applicants for driving licenses with a history of drug use. The sampling protocol consisted of collection of one hair (sample A, 5-cm length) and one urine sample. When hair and urine (EMIT Syva, cut-off levels: 0.3 mg/l for opiates, 0.15 mg/l for cocaine, GC-MS confirmation of positives) were both positive or negative the protocol was concluded. In the other cases, the assessment of 'current exposure' to drugs was carried out, in order to avoid seriated random urinalysis, by collecting a second hair sample (sample B) 6 weeks later and analysing the proximal 1-cm segment. Out of the 214 'A' hair samples analyzed, 14 (6.5%) tested positive for morphine and/or 6-acetylmorphine (6AM), and 26 (12%) for cocaine and/or benzoylecgonine (BE), whereas none of the samples tested positive for both drugs. Levels between 0.1 and 1 ng/mg of the single analytes were found in eight out of the 14 morphine-6AM positives (57%) and in 18 out of the 26 cocaine-BE positives (69%). The time course of positive cases showed a progressive decrease of morphine-6AM positives and a corresponding increase of cocaine-BE positives within the study period September 1995-February 1999. No cases with positive urine and negative hair were observed. Among the 40 positive cases, seven (four and three for opiates and cocaine, respectively) were found to be 'currently exposed to drug', four by urinalysis (three and one) and three by analysis of the hair sample B (1 and 2).

Automobile Driving↗
PubMed2000

Co-existence of cutaneous T-cell lymphoma and B hairy cell leukemia.

A primary cutaneous form of peripheral T-cell lymphoma (PTCL) and a low grade B-cell non-Hodgkin's lymphoma that was classified as a variant of hairy cell leukemia (HCL) were simultaneously diagnosed in a 79-year-old woman by both phenotypic and genotypic analyses. The coexistence of a T- and B-cell lymphoma in the same patient is rare, and, to our knowledge, this particular association has not been previously described. The patient was referred to our Department for evaluation of multiple cutaneous itchy, reddish plaques; laboratory analyses disclosed a lymphocytosis, that presented 6 years earlier. A bone marrow aspirate showed a 50% B-cell interstitial infiltrate, while a skin biopsy surprisingly revealed a PTCL. Clonality of both neoplastic processes was assessed by Southern blot analysis. The indolent clinical course of the cutaneous disease, and the low and stable number of circulating neoplastic T cells supported the diagnosis of a mycosis fungoides (MF)-like PTCL. Possible oncogenic events and/or putative underlying viral infections which could have played a role in the occurrence of B- and T-cell non-Hodgkin's lymphomas in the same patient are discussed.

Aged↗
PubMed2000

Anomalous transcripts and allelic deletions of the FHIT gene in human esophageal cancer.

The fragile histidine triad (FHIT) gene is localized on chromosome 3p14 and spans the common fragile site FRA3B. Even though its role in carcinogenesis is still unclear, this gene is frequently inactivated by carcinogen-induced intragenic deletions in many types of cancers, and FHIT abnormal transcripts are found in many primary tumors and tumor-derived cell lines. We evaluated FHIT gene involvement in 39 esophageal carcinomas (18 adenocarcinomas [AC¿, 21 squamous cell carcinomas [SCC]) by both reverse transcriptase-polymerase chain reaction (RT-PCR) amplification and loss of heterozygosity analysis (LOH). Thirty cases (77%) displayed either aberrant FHIT transcripts (12 cases) and/or LOH (24 cases); among these, only 6 samples displayed both aberrant transcripts and LOH, thus suggesting that the two events are probably independent. Moreover, LOH was significantly higher in SCC (80%) than in AC (44%), and because most of our patients are heavy smokers and/or alcohol consumers, these results suggest that the FHIT gene might be a common target for carcinogens also in the esophagus.

Acid Anhydride Hydrolases↗
PubMed2000

Lymphangioma of the spleen in an elderly patient.

Splenic cystic lymphangioma is a very rare condition, and is classified among cystic proliferations of the spleen. It is considered to be the result of a developmental malformation of the lymphatic system and can involve the spleen alone or be a part of multiorgan disease. It is usually seen in children, often found incidentally. We describe a case of cystic lymphangioma of the spleen in an elderly woman putting emphasis on the rarity of the case in old age, and on the problems of differential diagnosis with the other cystic proliferations of the spleen, in particular hydatid disease, in the absence of histologic information.

Aged↗
PubMed1999

Identification of a 3 kb Alu-mediated BRCA1 gene rearrangement in two breast/ovarian cancer families.

Most of the hereditary breast cancers are attributed to constitutive alterations of either BRCA1 or BRCA2 genes; nonetheless, germline mutations of these genes in 'high risk' families are found less frequently than expected from linkage data. Recent findings suggest that major genomic rearrangements of the BRCA1 gene might account for at least some of the apparently mutation negative cases. We studied 60 affected probands belonging to families with a strong history of breast and/or ovarian cancer who scored negative for BRCA1 gene mutations by PTT and SSCP analysis. DNA was analysed by the Southern blotting procedure using three different restriction enzymes, and two probes obtained by RT-PCR of the 5' and 3' BRCA1 coding sequence. A 3 kb deletion encompassing exon 17 and causing a frameshift mutation was identified in two independently ascertained families. RT-PCR and long-range DNA PCR were employed to characterize the rearrangement that was finally shown to be the result of a recombination between two very similar Alu repeats. This type of mutation is not identified by the conventional methods of mutation detection which are based on PCR amplification of single exons. Therefore, further search for gene rearrangements is needed to better define the proportion of 'high risk' families that might be explained by gross genomic alterations of the BRCA1 gene.

Alu Elements↗
PubMed1999

Immunological screening of drugs of abuse and gas chromatographic-mass spectrometric confirmation of opiates and cocaine in hair.

The work presents an analytical strategy to detect drugs of abuse in hair. It involves two sequential steps: a screening by a simple enzyme-linked immunosorbent assay (ELISA) methodology to detect opiates, cocaine and its metabolites, and benzodiacepines, followed by confirmation of opiates and cocaine metabolites in positive samples by gas chromatography coupled to mass spectrometry (GC-MS). In the same GC-MS run other drugs for substitution therapy (e.g. methadone and its main metabolite) can also be detected. After a double washing of hair samples with dichloromethane, hair specimens were cut into small pieces and 10 mg samples were incubated in 2 ml of methanol-trifluoroacetic acid (9:1) mixture, overnight at 37 degrees C. Aliquots of the extract were then evaporated, reconstituted in buffer and analysed according to the ELISA procedure. Confirmation involved solid-phase extraction of another fraction of the extract kept at -20 degrees C, derivatization with heptafluorobutyric anhydride and hexafluoroisopropanol and detection of cocaine, benzoylecgonine, ecgonine methylester, cocaethylene, morphine, codeine, 6-monoacetylmorphine, methadone and 2-ethylidene-1.5-dimethyl-3,3-diphenylpirrolidine (methadone metabolite) by selective ion monitoring after gas chromatographic separation. During the development of the method it was verified that no more than 10% of cocaine, opiates and benzodiacepines were lost when dichloromethane was used to wash real samples. The results also confirmed the increase of extractability power of TFA when it was added to methanol: the recovery for the analytes (cocaine and its metabolites and opiates) added to methanol-TFA alone was of the order of 90% except for benzoylecgonine (75%), and the recovery for the analytes added to methanol-TFA extract of drug-free hair was about 90% for all analytes except for benzoylecgonine and 6-MAM (around 70%). Regarding the stability of labile compounds, only small amounts of ecgonine methylester (2.3%) and morphine (7.2%) were produced, from cocaine and 6-MAM respectively, after the whole extraction procedure and two weeks of storage of methanol-TFA extracts at -20 degrees C. Satisfactory results were obtained when the procedures were applied to the analysis of external proficiency testing hair samples and actual specimens from drug addicts.

Cocaine↗
PubMed1998

Development of a coupled-column liquid chromatographic-tandem mass spectrometric method for the direct determination of betamethasone in urine.

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS-MS), coupled-column LC (LC-LC) combined with single quadrupole MS, and LC-LC-MS-MS. Both the three-step configurations (LC-MS-MS and LC-LC-MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC-MS-MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC-LC-MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC-LC-MS-MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h(-1)), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.

Betamethasone↗
PubMed1998

Fully-automated systematic toxicological analysis of drugs, poisons, and metabolites in whole blood, urine, and plasma by gas chromatography-full scan mass spectrometry.

The availability of automated, rapid and reliable methods for the systematic toxicological analysis (STA) of drugs and poisons in biosamples is of great importance in clinical and forensic toxicology laboratories. Gas chromatography-continuous scan mass spectrometry (GC-MS) possesses a high potential in STA because of its selectivity and identification power. However, in order to develop a fully automated STA method based on GC-MS two main obstacles have to be overcome: (a) sample preparation is rather sophisticated owing to the need to isolate analytes from the aqueous matrix and to allow a correct GC repartition of polar analytes; (b) the large amount of information collected within a single analysis makes it difficult to isolate relevant analytical information (mass spectra of analytes) from the chemical noise. Using a bench-top GC-MS system equipped with a laboratory robot for sample preparation (the Hewlett-Packard 7686 PrepStation) and an original method for mass spectral purification, a fully automated STA procedure was developed involving isolation of drugs from the sample (whole blood with minimal pretreatment, plasma, urine) by means of solid-phase extraction, derivatization (trimethylsilylation) of the acidic-neutral and of the basic extracts, GC-MS analysis, processing of data, and reporting of results. Each step of the procedure, and the method for data analysis in particular, can be easily integrated with other existing STA methods based on GC-MS.

Autoanalysis↗