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Statins are cholesterol-lowering medications which are widely prescribed as first-line treatment for hyperlipidemia, against high blood cholesterol aimed at reducing the risk of atherosclerotic diseases. Notwithstanding their undoubted efficacy, the needed long-term treatment with these drugs is characterized by a high percentage of dropout. Consequently, an effective tool to verify the patients' compliance to statin therapy is needed. In this context, the analysis for drugs and drug metabolites in the hair may represent an almost ideal tool because, according to a sound body of forensic toxicological literature, concentrations in the hair matrix reflect the chronic intake of drugs and pharmaceuticals. In this light, in the present study, a novel, specific and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method has been developed to determine six statins and their metabolites (namely atorvastatin, (p)α-OH-atorvastatin-lactone, (o)α-OH-atorvastatin-lactone, rosuvastatin, N-desmethyl rosuvastatin and pravastatin) in human hair. After optimization, the method was successfully validated in terms of selectivity, linearity, sensitivity, precision, accuracy, stability and matrix effect. Moreover, the practical applicability of this method for verifying adherence to statin therapy was assessed by testing samples of hair collected from subjects under long-term therapy with statins.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Atorvastatina/uso terapéutico , Análisis de Cabello , Pravastatina/uso terapéutico , ColesterolRESUMEN
INTRODUCTION: The estimation of post-mortem interval (PMI) remains a major challenge in forensic science. Most of the proposed approaches lack the reliability required to meet the rigorous forensic standards. OBJECTIVES: We applied 1H NMR metabolomics to estimate PMI on ovine vitreous humour comparing the results with the actual scientific gold standard, namely vitreous potassium concentrations. METHODS: Vitreous humour samples were collected in a time frame ranging from 6 to 86 h after death. Experiments were performed by using 1H NMR metabolomics and ion capillary analysis. Data were submitted to multivariate statistical data analysis. RESULTS: A multivariate calibration model was built to estimate PMI based on 47 vitreous humour samples. The model was validated with an independent test set of 24 samples, obtaining a prediction error on the entire range of 6.9 h for PMI < 24 h, 7.4 h for PMI between 24 and 48 h, and 10.3 h for PMI > 48 h. Time-related modifications of the 1H NMR vitreous metabolomic profile could predict PMI better than potassium up to 48 h after death, whilst a combination of the two is better than the single approach for higher PMI estimation. CONCLUSION: The present study, although in a proof-of-concept animal model, shows that vitreous metabolomics can be a powerful tool to predict PMI providing a more accurate estimation compared to the widely studied approach based on vitreous potassium concentrations.
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Cambios Post Mortem , Potasio , Ovinos , Animales , Potasio/análisis , Cuerpo Vítreo/química , Reproducibilidad de los Resultados , MetabolómicaRESUMEN
Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays-based methods are generally employed but digoxin- and digitoxin-like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC-MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision, and recovery. A simple liquid-liquid extraction procedure was used for sample clean-up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC-MS/MS method versus the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross-reaction events with endogenous digoxin-like substances.
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Digitoxina , Digoxina , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida , Digitoxina/química , Digoxina/química , Inmunoensayo , Espectrometría de Masas en Tándem/métodosRESUMEN
The continuous introduction in the market of new psychoactive drugs (NPS) represents a well-known international emergency. Indeed, the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime are paying great attention to the spread of NPS. In addition to the traditional analytical approaches based on GC-MS and HPLC-MS, also CE coupled with MS has proved to be a precious tool for the toxicological screening of biosamples. On these grounds, the aim of the present work was to test the application of CE-HRMS as a new screening tool for the rapid detection of these novel drugs in urine. Separations were performed in an uncoated fused-silica capillary with id of 75 µm with a total length of 100 cm, by applying a constant voltage of 15 kV. The QTOF-MS was implemented with an electrospray ion source operating in positive ionization full scan mode in the range of 100-1000 m/z. Under these conditions, different NPS has been tested, including eight cathinones, five phenethylamine, and seven tryptamines. The method was validated after optimization of the following analytical parameters: BGE composition and pH, separation voltage, sheath liquid composition, and flow rate and ESI source settings. The applicability of the method was successfully tested by analyzing a series of real urine samples obtained from drug users.
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Alcaloides/orina , Aminas/orina , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Psicotrópicos/orina , Humanos , Límite de Detección , Modelos Lineales , Reproducibilidad de los Resultados , Detección de Abuso de SustanciasRESUMEN
Abusive head trauma (AHT) in children is notoriously one of the most challenging diagnoses for the forensic pathologist. The pathological "triad", a combination of intracranial subdural haematoma, cerebral oedema with hypoxic-ischaemic changes and retinal haemorrhages, is frequently argued to be insufficient to support a corroborated verdict of abuse. Data from all available English-language scientific literature involving radiological and neuropathological spinal cord examination is reviewed here in order to assess the contribution of spinal cord changes in differentiating abusive from accidental head trauma. In agreement with the statistically proven association between spinal subdural haemorrhage (SDH) and abuse (Choudhary et al. in Radiology 262:216-223, 2012), spinal blood collection proved to be the most indicative finding related to abusive aetiology. The incidence of spinal blood collection is as much as 44-48% when all the spinal cord levels are analysed as opposed to just 0-18% when the assessment is performed at cervical level only, in agreement with the evidence of the most frequent spinal SDH location at thoracolumbar rather than cervical level. In this review, the source of spinal cord blood collection and how the age of the child relates to the position of spinal cord lesions is also discussed. We concluded that the ante mortem MRI examination and post mortem examination of whole-length spinal cord is of fundamental interest for the assessment of abuse in the forensic setting.
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Maltrato a los Niños/diagnóstico , Traumatismos Craneocerebrales/sangre , Traumatismos Craneocerebrales/patología , Patologia Forense , Traumatismos de la Médula Espinal/sangre , Traumatismos de la Médula Espinal/patología , Factores de Edad , Niño , Preescolar , Humanos , LactanteRESUMEN
Estimation of the post-mortem interval (PMI) remains a matter of concern in the forensic scenario. Traditional and novel approaches are not yet able to fully address this issue, which relies on complex biological phenomena triggered by death. For this purpose, eye compartments may be chosen for experimental studies because they are more resistant to post-mortem modifications. Vitreous humour, in particular, has been extensively investigated, with potassium concentration ([K+]) being the marker that is better correlated with PMI estimation. Recently, a 1H nuclear magnetic resonance (NMR) metabolomic approach based on aqueous humour (AH) from an animal model was proposed for PMI estimation, resulting in a robust and validated regression model. Here we studied the variation in [K+] in the same experimental setup. [K+] was determined through capillary ion analysis (CIA) and a regression analysis was performed. Moreover, it was investigated whether the PMI information related to potassium could improve the metabolome predictive power in estimating the PMI. Interestingly, we found that a part of the metabolomic profile is able to explain most of the information carried by potassium, suggesting that the rise in both potassium and metabolite concentrations relies on a similar biological mechanism. In the first 24-h PMI window, the AH metabolomic profile shows greater predictive power than [K+] behaviour, suggesting its potential use as an additional tool for estimating the time since death.
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Humor Acuoso/química , Metabolómica , Cambios Post Mortem , Potasio/análisis , Animales , Modelos Animales de Enfermedad , Electroforesis Capilar , Espectroscopía de Protones por Resonancia Magnética , Análisis de Regresión , OvinosRESUMEN
Forensic inquests, particularly, in assessing time since death currently recognize the importance of the analysis of vitreous humour (VH) biomarkers. Present research, studies, and validates the determination of lactate (La) in VH by CZE with indirect UV detection. The BGE (pH 8.9) consisted of Tris buffer (37 mM) containing 4-methoxybenzoic acid (4 mM) and alkyl-trimethyl-ammonium bromide (1.2 mM). Each VH specimen was diluted with a butyric acid solution (internal standard 0.057 mM) and La and butyrate were separated within 3-5 min (30 kV). The La LOQ and LOD were 4 and 2 mM, respectively. The calibration curve linearity ranged from 4 to 80 mM; intra- and interruns precisions were less than 10% for standard as well as for VH specimen, respectively. To investigate postmortem interval (PMI) and VH lactate level correlation, human VH specimens were collected during autopsy (n = 40) and stored at -20°C until assay. La levels ranged from 16 to 42 mM; PMI values ranged from 10 to 141 h. La (mM) and PMI (h) correlation was statistically significant (r2 = 0.527; p < 0.05). In conclusion, the present CZE analysis is efficacious to determine VH La as a biomarker for PMI investigation.
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Electroforesis Capilar/métodos , Medicina Legal/métodos , Ácido Láctico/análisis , Cuerpo Vítreo/química , Adulto , Anciano , Anciano de 80 o más Años , Autopsia , Femenino , Humanos , Límite de Detección , Modelos Lineales , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
The phosphoinositide (PI) signal transduction pathway participates in liver metabolism. Abnormal activity or expression of PI-specific phospholipase C (PLC) enzymes has been described in different liver diseases. We resume the role of the PI metabolism in liver and PLC abnormalities in different liver diseases. Moreover, we present the results of PLC analyses in a normal human liver and an alcohol-damaged liver. PLC enzymes and the expression of the corresponding genes in liver biopsies from individuals deceased for complications of the alcoholic liver disease (ALD) at different stages compared with normal controls (deceased individuals with histologically normal livers without alcohol addiction anamnesis) were analyzed by using immunohistochemistry and molecular biology techniques. The expression panel of PLCs was described in normal and alcohol abuse liver. Our observations suggest that the regulation of PLC expression might be due to posttranscriptional events and that alcohol affects the epigenetic control of PLC expression belonging to PI signaling. We also describe the alternate expression of PLCB1 and PLCH1 genes in liver. Our results corroborate literature data suggesting that PLC enzymes are differently expressed in normal versus pathological liver, playing a role in the histopathogenesis of liver tissue damage. The expression and/or localization of selected PLC isoforms is especially affected in alcohol-related liver tissue histopathology. Our present observations confirm that the modulation of protein synthesis plays a role in the regulation of PLC enzymes. We also suggest that this modulation might act at the transcription level. Further studies are required to investigate related epigenetic mechanisms.
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BACKGROUND: Erythrocyte mean corpuscular volume (MCV) has been used for decades as a biomarker of chronic alcohol abuse and in the treatment of alcohol dependence. More recently, it has also been adopted to investigate the fitness of subjects to hold the driving license to prevent traffic accidents. So far, however, the studies on the association of MCV with an increased risk of alcohol-associated car accidents are extremely scarce, if not totally absent. To the best of our knowledge, the present work is the first specifically aimed at studying a plausible association between elevated MCV and crash accidents correlated with alcohol abuse. METHODS: A total of 6,244 drivers involved in traffic accidents underwent mandatory laboratory analyses including blood alcohol concentration (BAC) determination and MCV analysis. BAC and MCV determinations were performed by headspace gas chromatography and complete blood count, respectively. RESULTS: The chi-square test evaluating the proportions of subjects with elevated MCVs (>95 fl) yielded a highly significant result (χ2 = 68.0; p < 0.001) in the blood samples where the BAC was above the legal limit (i.e., >0.5 g/l). However, when considering only drivers showing BACs in the range of 0.51 to 1.5 g/l, the frequencies of elevated MCV values are fairly comparable (χ2 = 0.062, p = 0.80). In contrast, limiting the evaluation to BACs > 1.5 g/l, the frequency of elevated MCVs raised to 19.1% (χ2 = 58.9, p value < 0.001 vs. the group with BAC within the legal limits). CONCLUSIONS: The present observations show that MCV increases are typically associated with drivers involved in accidents only if driving under severe alcohol intoxication, leading to a preliminary conclusion that, in the context of the certification of the fitness to the driving license, MCV fails to reveal individuals at risk who tend to drive in a condition of low-to-moderate alcohol intoxication.
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Accidentes de Tránsito/estadística & datos numéricos , Alcoholismo/sangre , Conducir bajo la Influencia , Índices de Eritrocitos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Intoxicación Alcohólica/sangre , Intoxicación Alcohólica/psicología , Conducción de Automóvil/legislación & jurisprudencia , Conducción de Automóvil/psicología , Biomarcadores , Recuento de Células Sanguíneas , Depresores del Sistema Nervioso Central/sangre , Cromatografía de Gases , Etanol/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
Background Although the post-mortem increase of ammonium in biological fluids is well known, ammonium analysis in vitreous humour has never been used in recent times for the determination of the post-mortem interval. The present work represents a new application of capillary electrophoresis with indirect UV detection in the field of forensic analysis. Methods The electrophoretic separation was carried out in a running buffer made of 5 mM imidazole, 5 mM 18-crown-6 ether and 6 mM d,l-α-hydroxybutyric acid (HIBA). To overcome the lack of optical absorption of ammonium, indirect UV detection was applied. The used wavelength was 214 nm. Results The method showed good linearity in the concentration range from 0.16 to 5.0 mM. The limit of detection, 0.039 mmol/L, was established on the basis of the linearity curve. Precision and bias studies carried out on the pure ammonium solutions and in real biological samples, revealed %RSDs well below 20%. A preliminary application to real cases where the death time was precisely known (14 bodies) was carried out plotting vitreous humour ammonium vs. post-mortem interval with a resulting good linear correlation until 100 h post-mortem. Conclusions After validation in real cases, the present method can become a powerful tool to unravel one of the most challenging issues of forensic investigation: determination of the time of death.
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Compuestos de Amonio/análisis , Electroforesis Capilar/métodos , Medicina Legal , Espectrofotometría Ultravioleta/métodos , Cuerpo Vítreo/química , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVE: The study compare two tests for evaluating the driving abilities of patients undergoing opioid therapy for chronic pain: the Vienna Test System (VTS), a software developed for this purpose, and a new free APP for smartphones (SafeDrive) measuring visual and auditory reaction times. METHODS: One hundred and five patients undergoing long term opioid therapy for chronic pain were enrolled. The driving abilities of study patients were evaluated using two tests, namely the Vienna test System (VTS) and the SafeDrive APP. The concordance between the two tests was evaluated through Cohen's test. In addition we evaluated the correlation between the results of both VTS and SafeDrive tests and prescribed Morphine Equivalent Doses (MEDs), sex, age and the specific drugs taken, by multivariate linear regression analysis. RESULTS: A statistically significant concordance (Cohen's K coefficient=0.476) was found between the SafeDrive APP and the VTS; multivariate linear regression analysis found no significant influences of dosage and type of opioid prescribed on test performances, but significant influences of sex and age. CONCLUSIONS: The Authors found a significant correlation between VTS with SafeDrive test results. The SafeDrive APP is cheaper, easier to use and faster than VTS, and is portable and "usable on the road". Complex behavioral tasks such as driving may be severely impaired by psychoactive drugs, and consequently SafeDrive could be considered a useful portable screening tool to identify drivers with drug associated psychomotor impairment.
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Analgésicos Opioides/uso terapéutico , Conducción de Automóvil , Dolor Crónico/tratamiento farmacológico , Destreza Motora/efectos de los fármacos , Humanos , Teléfono InteligenteRESUMEN
The polysialylated isoform of the neural cell adhesion molecule (PSA-NCAM) has been shown to be a key player in neuroplastic changes and is expressed in various disorders. We investigated the PSA-NCAM expression on brain cortical tissue in a cohort of drug-related deaths. Brains from 25 drug abusers and 10 control subjects were removed at autopsy, and 2 samples of the right parietal lobe of each case were obtained. The polysialylated isoform of NCAM was evaluated on formalin-fixed and paraffin-embedded tissues. Eleven patients were polydrug abusers; 14 used a single substance. The mechanisms of death were acute respiratory failure (n = 19), cardiorespiratory failure (n = 4), acute heart failure (n = 1), and brain injury (n = 1). Toxicological analyses of blood were available for all cases, and urine and bile analyses for 19 of 25 cases. The polysialylated isoform of NCAM immunoexpression in the neuronal soma and dendritic spines was observed in 18 (72%) of 25 drug abusers and in 2 (20%) of 10 control subjects. Drug abusers were statistically more positive for PSA-NCAM than control subjects (P = 0.0082). The expression of PSA-NCAM in the parietal cortex could be an indicator of brain damage due to drug abuse, and its availability could allow the forensic pathologists to develop rapid and low-cost additional or alternative method to improve detection of drug-related deaths.
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Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Lóbulo Parietal/metabolismo , Ácidos Siálicos/metabolismo , Trastornos Relacionados con Sustancias/diagnóstico , Adulto , Biomarcadores/metabolismo , Daño Encefálico Crónico/diagnóstico , Daño Encefálico Crónico/metabolismo , Estudios de Casos y Controles , Femenino , Patologia Forense , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/metabolismo , Trastornos Relacionados con Sustancias/metabolismo , Adulto JovenRESUMEN
In this study, DNA extracted from known buccal samples was combined into two component mixture samples. These were subjected to UV exposure prior to their amplification with the Promega PowerPlex® 16HS amplification kit, and subsequent capillary electrophoresis on the ABI 3130xl instrument. Damaged samples were subjected to enzymatic repair treatment and retested to assess the amount of repair. Data showed that there is fidelity associated with the application with profile concordance after its use, and a corresponding increase in the amount of recovered alleles post damage. Results also showed changes in the stochastic relationship between mixture components that appear to be induced by the repair process itself. The mixture ratios of DNA samples were altered from an approximate original 1:3 ratio, to a ratio of 1:2 or greater. This variation can have a significant effect regarding the ability to reliably de-convolute DNA mixtures that have been subjected to the repair process.
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Dermatoglifia del ADN , Reparación del ADN , ADN/genética , HumanosRESUMEN
This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.
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Líquidos Corporales/química , Cromatografía Líquida de Alta Presión/métodos , Colorantes Fluorescentes/química , Terbio/química , Transferrina/análogos & derivados , Humanos , Espectrometría de Fluorescencia , Transferrina/análisisRESUMEN
Transferrin (Tf) is the major iron-transporting protein in the human body and, for this reason, has been extensively studied in biomedicine. This protein undergoes a complex glycosylation process leading to several glycoforms, some of which are important in the diagnosis of alcohol abuse and of congenital glycosylation defects under the collective name of carbohydrate-deficient transferrin (CDT). Exploiting the Tf ability to bind not only iron but also other ions, specific attention has been devoted to binding activity towards Tb3+, which was reported to greatly enhance its intrinsic fluorescence upon the interaction with Tf. However, the structural properties of the Tb3+-Tf complex have not been described so far. In the present work, the formation of the Tf-Tb3+ complex has been investigated by the employment of several biophysical techniques, such as fluorescence resonance energy transfer (FRET), "native" mass spectrometry (MS), and near-UV circular dichroism (CD). Each method allowed the detection of the Tf-Tb3+ complex, yielding a specific signature. The interaction of Tb3+ with Fe3+-free Tf (apoTf) has been described in terms of stoichiometry, affinity, and structural effects in comparison with Fe3+. These experiments led to the first direct detection of the Tf-Tb3+ complex by MS, indicating a 1:2 stoichiometry and allowing the investigation of structural effects of metal binding. Either Tb3+ or Fe3+ binding affected protein conformation, inducing structural compaction to a similar extent. Nevertheless, near-UV CD and pH-dependence profiles suggested subtle differences in the coordination of the two metals by Tf side chains. Experimental conditions that promote complex formation have been identified, highlighting the importance of alkaline pH and synergistic ions, such as carbonate. On the basis of these studies, sample pretreatment, separation, and detection conditions of a high-performance liquid chromatographic method for CDT analysis are optimized, achieving relevant increase (by a factor of â¼3) of analytical sensitivity. Graphical abstract Schematic representation of HPLC-separated transferrin glycoforms detected by fluorescence emission of the terbium ions bound to the protein.
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Cromatografía Líquida de Alta Presión/métodos , Complejos de Coordinación/química , Colorantes Fluorescentes/química , Terbio/química , Transferrina/análogos & derivados , Quelantes/química , Dicroismo Circular , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Espectrometría de Masas , Modelos Moleculares , Conformación Proteica , Transferrina/químicaRESUMEN
BACKGROUND: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal. METHODS: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material. RESULTS: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%. CONCLUSIONS: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results.
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Servicios de Laboratorio Clínico/normas , Hepcidinas/sangre , Cooperación Internacional , Humanos , Inmunoquímica , Modelos Lineales , Estándares de ReferenciaRESUMEN
Continued progress in chronic alcohol abuse investigation requires the development of less invasive procedures for screening purposes. The application of finger-prick and related dried blood spots (fpDBS) for carbohydrate deficient transferrin (CDT) detection appears suitable for this aim. Therefore, the goal of this project was to develop a screening method for CDT using fpDBS with CZE analysis. Blood samples prepared by finger-prick were placed on DBS cards and left to air dry; each dried fpDBS disc was shredded into small pieces and suspended in acid solution (60 µL of HCl 120 mmol/L). After centrifugation (10 min at 1500 × g), the collected sample was adjusted to pH 3.5. After an overnight incubation, the pH was neutralised and an iron rich solution was added. After 1 h, CZE analysis was carried out. A group of 47 individuals was studied. Parallel serum samples were collected from each investigated subject and the %CDT for each sample was measured using HPLC and CZE techniques. The fpDBS transferrin sialo isoform electropherograms were similar to those obtained with serum. Moreover, fpDBS CZE CDT percentage levels demonstrated significant statistical correlation with those obtained from serum for both HPLC and CZE %CDT (p < 0.01; r2 = 0.8913 and 0.8976, respectively), with %CDT from 0.8 to 13.7% for fpDBS and from 0.7 to 12.7% for serum. The newly developed fpDBS procedure for CDT analysis provides a simple and inexpensive tool for use in population screening.
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Pruebas con Sangre Seca/métodos , Electroforesis Capilar/métodos , Toxicología Forense/métodos , Transferrina/análogos & derivados , Adulto , Anciano , Alcoholismo/sangre , Alcoholismo/diagnóstico , Cromatografía Líquida de Alta Presión/métodos , Femenino , Humanos , Límite de Detección , Modelos Lineales , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Transferrina/análisisRESUMEN
In response to the need for straightforward analytical methods to assess the affinity of molecularly imprinted nanoparticles (MIP NPs) for ligands, capillary electrophoresis (CE) was exploited using MIP NPs targeting the iron-regulating hormone hepcidin. In this work, MIP NPs were challenged with their template peptide, i.e., the N-terminal 5-mer of hepcidin, in comparison to unrelated ligand peptides. A CE separation method was developed ex novo achieving, after optimization of the background electrolyte (150 mM sodium phosphate pH 7.4) and of the running temperature (35 °C), the full separation of the free ligand from the complexed MIP NPs. The CE binding isotherm allowed the estimation of a micromolar dissociation constant for the 5-mer template-MIP NPs complex, in agreement with independent measurements. The CE offered the advantages of a direct injection of the MIP NPs/ligand incubation mix, without preliminary fractionation steps, requiring only minimal sample volumes and short analysis times. In conclusion CE proved to be a valid technique for characterizing the interactions of MIP NP libraries for selected target compounds.
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Electroforesis Capilar/métodos , Impresión Molecular/métodos , Nanopartículas , Sitios de Unión , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Ligandos , TemperaturaRESUMEN
OBJECTIVE: To analyze the morphology and depth of stab wounds experimentally produced on human legs amputated for medical reasons using multislice computed tomography (MSCT) and magnetic resonance imaging (MRI) after the instillation of a single contrast medium solution (CMS). MATERIALS AND METHODS: For morphological analysis, MSCT and MRI scans were performed before and after the instillation of CMS into the wound cavity. Depth measurements were performed on the sagittal view only after CMS instillation. Subsequently, each wound was dissected using the layer-by-layer technique and the depth was measured by a ruler. One-way between-groups pairwise analysis of variance (ANOVA) and Bland-Altman plot analysis were used for comparing radiological and anatomical measurements. RESULTS: Unenhanced MSCT images did not identify the wound channels, whereas unenhanced MRI evidenced the wound cavity in 50 % of cases. After the instillation of CMS, both MSCT and MRI depicted the wound channel in all the investigated stabbings, although the morphology of the cavity was irregular and did not resemble the shape of the blade. The radiological measurements of the wounds' depth, after the application of CMS, exhibited a high level of agreement (about 95 % at Bland-Altman plot analysis) with the anatomical measurements at dissection. A similar systematic underestimation, however, has been evidenced for MSCT (average 11.4 %; 95 % CI 7-17) and MRI (average 9.6 %; 95 % CI 6-13) data after the instillation of CMS with respect to wound dissection measurements. CONCLUSION: MSCT and MRI after the instillation of CMS can be used for depicting the morphometric features of stab wounds, although depth measurements are affected by a slight systematic underestimation compared to layer-by-layer dissection.
Asunto(s)
Medicina Legal/métodos , Traumatismos de la Pierna/diagnóstico por imagen , Imagen por Resonancia Magnética , Tomografía Computarizada Multidetector , Heridas Punzantes/diagnóstico por imagen , Medios de Contraste , Humanos , Técnicas In VitroRESUMEN
BACKGROUND: Carbohydrate-deficient transferrin (CDT) is a well-recognized highly specific marker of chronic alcohol abuse. The association of CDT with alcohol-related traffic accidents was evaluated to objectively validate the use of this marker for certifying the physical fitness for driving license regranting after its confiscation for drunk driving. METHODS: The study was carried out on 468 injured drivers (InjDr), who underwent mandatory blood alcohol concentration (BAC) and drug analysis in biological fluids. The InjDr group was divided into 2 subgroups on the basis of BAC legal limit adopted in Italy (BAC ≤ 0.5 g/l: InjDr1 ; BAC >0.5 g/l: InjDr2 ). The control group (CntDr) included 236 subjects holding safety-sensitive job positions and undergoing mandatory toxicological analyses. The determination of BAC in blood and CDT in serum were performed using validated analytical methods based on head-space gas chromatography and high-performance liquid chromatography, respectively. RESULTS: The evaluation of CDT distribution in the 3 groups (CntDr, InjDr1 , InjDr2 ) showed that CDT distribution in the InjDr1 group was similar to that observed in the CntDr group (p = 0.159) and different from that observed in the InjDr2 group (p < 0.001). Partitioning the CDT data of each group into "CDT positives" and "CDT negatives" on the basis of the cut off (1.90%), it was possible to calculate the odds of the 3 groups and then the odds ratios. The odds ratio of InjDr1 versus CntDr was 4.56 (p = 0.158), whereas the odds ratio of InjDr2 versus CntDr was 132 (p < 0.001). Furthermore, a dose-response effect was found only when comparing InjDr2 with CntDr. CONCLUSIONS: The data of the present study strongly support the use of the CDT test to evaluate the risk of a subject to be involved in a road accident while driving under the influence of alcohol.