Alessandro Nalesso

Shortened telomeres in individuals with abuse in alcohol consumption

Alcohol abuse leads to earlier onset of aging‐related diseases, including cancer at multiple sites. Shorter telomere length (TL) in peripheral blood leucocytes (PBLs), a marker of biological aging, has been associated with alcohol‐related cancer risks. Whether alcohol abusers exhibit accelerated biological aging, as reflected in PBL‐TL, has never been examined. To investigated the effect of alcohol abuse on PBL‐TL and its interaction with alcohol metabolic genotypes, we examined 200 drunk‐driving traffic offenders diagnosed as alcohol abusers as per the Diagnostic and Statistical Manual of Mental Disorders [DSM‐IV‐TR] and enrolled in a probation program, and 257 social drinkers (controls). We assessed alcohol intake using self‐reported drink‐units/day and conventional alcohol abuse biomarkers (serum γ‐glutamyltrasferase [GGT] and mean corpuscular volume of erythrocytes [MCV]). We used multivariable models to compute TL geometric means (GM) adjusted for age, smoking, BMI, diet, job at elevated risk of accident, genotoxic exposures. TL was nearly halved in alcohol abusers compared with controls (GMs 0.42 vs. 0.87 relative T/S ratio; p < 0.0001) and decreased in relation with increasing drink‐units/day (p‐trend = 0.003). Individuals drinking >4 drink‐units/day had substantially shorter TL than those drinking ≤4 drink‐units/day (GMs 0.48 vs. 0.61 T/S, p = 0.002). Carriers of the common ADH1B*1/*1 (rs1229984) genotype were more likely to be abusers (p = 0.008), reported higher drink‐units/day (p = 0.0003), and exhibited shorter TL (p < 0.0001). The rs698 ADH1C and rs671 ALDH2 polymorphisms were not associated with TL. The decrease in PBL‐TL modulated by the alcohol metabolic genotype ADH1B*1/*1 may represent a novel mechanism potentially related to alcohol carcinogenesis in alcohol abusers.

Phosphatidylethanol in Blood as a Marker of Chronic Alcohol Use: A Systematic Review and Meta-Analysis

The present paper aims at a systematic review of the current knowledge on phosphatidylethanol (PEth) in blood as a direct marker of chronic alcohol use and abuse. In March 2012, the search through “MeSH” and “free-text” protocols in the databases Medline/PubMed, SCOPUS, Web of Science, and Ovid/Embase, combining the terms phosphatidylethanol and alcohol, provided 444 records, 58 of which fulfilled the inclusion criteria and were used to summarize the current evidence on the formation, distribution and degradation of PEth in human blood: (1), the presence and distribution of different PEth molecular species (2), the most diffused analytical methods devoted to PEth identification and quantization (3), the clinical efficiency of total PEth quantification as a marker of chronic excessive drinking (4), and the potential utility of this marker for identifying binge drinking behaviors (5). Twelve papers were included in the meta-analysis and the mean (M) and 95% confidence interval (CI) of total PEth concentrations in social drinkers (DAI ≤ 60 g/die; M = 0.288 μM; CI 0.208–0.367 μM) and heavy drinkers (DAI > 60 g/die; M = 3.897 μM; CI 2.404–5.391 μM) were calculated. The present analysis demonstrates a good clinical efficiency of PEth for detecting chronic heavy drinking.

High performance liquid chromatography–high resolution mass spectrometry and micropulverized extraction for the quantification of amphetamines, cocaine, opioids, benzodiazepines, antidepressants and hallucinogens in 2.5 mg hair samples

A high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method for simultaneous screening and quantification of 28 drugs was developed and validated for 2.5 mg hair samples. Target drugs and their metabolites included amphetamines, cocaine, opioids, benzodiazepines, antidepressants, and hallucinogens. After decontamination, hair samples were extracted with 200 μL of a mixture of water: acetonitrile:1 M trifluoroacetic acid (80:10:10, v/v) using a 5 min simultaneous pulverization/extraction step. The extracts were analysed by HPLC-HRMS in an Orbitrap at a nominal resolution of 60,000, with concomitant in source collisional experiments (in source CID). Gradient elution on an Atlantis T3 column resolved 28 target compounds and 5 internal standards. Total chromatographic run time was 26 min. Calibration was achieved by linear regression analysis utilizing six calibration points; R2 ranged from 0.9964 to 0.9999, the limits of quantification were 0.1 ng/mg for 8 compounds, 0.2 ng/mg for 16 compounds and 0.5 ng/mg for 4 compounds; mean relative errors from -21% to +23% were obtained; relative standard deviation, used to estimate repeatability and intermediate reproducibility at three concentrations, was always less than 20%. Process efficiency and recoveries for all analytes were better than 65 and 73%, respectively, at any concentration. The method was applied to hair samples from forensic investigations that contained a broad assortment of drugs of abuse and pharmaceuticals. The use of concomitant HRMS full scan and CID afforded the possibility of retrospective analysis for discovering untargeted drugs.

Quantitative profiling of phosphatidylethanol molecular species in human blood by liquid chromatography high resolution mass spectrometry

Phosphatidylethanol (PEth) is a group of aberrant phospholipids formed in cell membranes in the presence of ethanol by the catalytic action of the enzyme phospholipase D on phosphatidylcholine. Recently published literature has demonstrated the existence of several molecular species of PEth in samples drawn from alcohol-dependent subjects. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method coupled with a lipidomic strategy was developed and validated for the quantitative profiling of PEth molecular species in human blood collected from heavy and social drinkers. Chromatography was performed on a C18 column using acetonitrile, 10mM ammonium acetate, and 2-propanol as mobile phases with a 22-min gradient. HRMS experiments were performed on an LTQ-Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionization source operated in negative ion mode. The theoretical masses of [M-H](-) of PEth species were calculated from the elemental chemical formula by varying the length and unsaturation grade of the fatty acid side chains; identification of PEth species in blood was achieved by searching the accurate masses of the targeted compounds in the acquired full-scan LC-HRMS chromatogram. The chemical structure of tentatively identified PEth species was elucidated through HR multiple mass experiments. The validated LC-HRMS method was selective, as warranted by HRMS at 60,000 resolution and 4 ppm accuracy. Linearity was observed in the 0.001-2.000 μM range, and limit of detection of 0.0005 μM and limit of quantitation of 0.001 μM were obtained for single PEth species. Imprecision and inaccuracy were always lower than 10% and 15%, respectively. The identification capabilities of the method were tested on blood samples collected from heavy drinkers (n=11), social drinkers (n=8), and teetotalers (n=10). The high sensitivity of the method led to the simultaneous identification of 17 different PEth molecular species in blood collected from heavy drinkers, and 2 PEth species (16:0/18:1 and 16:0/18:2) in blood collected from social drinkers.

Determination of ketamine and norketamine in hair by micropulverized extraction and liquid chromatography–high resolution mass spectrometry

Ketamine (KT), primarily used as a general anaesthetic agent in clinical practice, has become very popular in recent years as a recreational drug, due to its dissociative and hallucinogenic effects. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method has been developed and validated for the quantification of KT and its main metabolite norketamine (NK) in 2.0mg of hair. Sample preparation consisted of a rapid, simultaneous pulverization and extraction step in acidic solution, followed by centrifugation and filtration. Gradient elution was performed by an Atlantis T3 analytical column, and deuterated KT was used as the internal standard. Positive ion electrospray ionization and HRMS determination in full-scan mode were achieved with an Orbitrap mass spectrometer. The method has a linear range of 0.05-50 ng/mg, a limit of quantisation of 0.05 ng/mg and a limit of detection of 0.02 ng/mg for both KT and NK. The validated method was applied for the determination of KT and NK in two authentic hair samples from subjects suspected of taking psychoactive substances. The detection of the metabolite at low concentration gave proof for systemic drug origin and an investigation into the possible presence of further metabolites was performed by means of retrospective screening.

Monitoring haloperidol exposure in body fluids and hair of children by liquid chromatography-high-resolution mass spectrometry.

Background: Haloperidol, 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone (HP), one of the most widely used antipsychotics in the treatment of schizophrenia, mania, and other psychiatric disorders, is frequently encountered in cases of unintentional pediatric intoxication because the ingestion of a small amount can cause significant toxic effects in children. For monitoring HP in suspected ingestions, a liquid chromatography–high-resolution mass spectrometry method has been developed and validated in urine, blood, and hair samples. Methods: The analyte was extracted from 1 mL blood or urine by liquid/liquid extraction and from 5 mg of hair by micropulverized extraction; gradient elution on an Atlantis T3 column was realized using HP-d4 as an internal standard. Positive ion electrospray ionization and high-resolution mass spectrometry determination were performed in an Orbitrap mass spectrometer. Results: The method exhibited a r2 > 0.999 in the studied ranges (0.1–50 ng/mL in urine and blood and 0.1–50 ng/mg in hair) and a limit of quantification of 0.1 ng/mL for urine and blood and 0.1 ng/mg for hair; intra-assay and interassay relative SDs were always more than 18%. The method was applied to determine haloperidol in 3 children who were admitted to emergency departments. HP concentrations ranged from 2 to 21 ng/mL in urine, from not detected to 4.9 ng/mL in blood, and from 0.37 to 0.73 ng/mg in hair samples. Conclusions: The utilization of high-resolution/high-accuracy mass spectrometry in full scan mode allowed the identification of HP metabolites in urine and blood, thus unequivocally documenting the exposure to the drug. HP metabolites were structurally characterized by high-resolution multiple mass spectrometry. For the first time, a HP metabolite was detected in hair.

Underlying Substance Abuse Problems in Drunk Drivers

Objectives: The aim of this study was to investigate polydrug use in drunk drivers. Methods: The experimental study was conducted on 2,072 drunk drivers undergoing a driving license reissue protocol at the Department of Legal Medicine of Padova University Hospital in the period between January 2011 and December 2012. The study protocol involved anamnesis, clinical examination, toxicological history, and toxicological analyses on multiple biological samples. Results: One thousand eight hundred seventy-seven subjects (90.6%) were assessed as fit to drive, and 195 (9.5%) were declared unfit. Among those unfit, 32 subjects (1.6%) were declared unfit due to recent use of an illicit drug (time span < 6 months), 23 (1.1%) spontaneously interrupted the protocol before its end, and 140 (6.8%) completed the assessment. Ineligibility to drive after completeness of the protocol was established in 1.2% of cases for alcohol disorders and in 5.7% of cases for illicit drug abuse; only one subject was included in both subgroups. Cocaine was the most widely used substance, followed by cannabis, opiates, and psychotropic pharmaceutical drugs. Conclusions: The application of the protocol presented in this study allowed the identification of underlying polydrug use in drunk drivers. The study led to the identification of 6.8% unfit subjects on the basis of alcohol disorders and/or drug abuse, compared to 1.2% of identifiable unfitness if the protocol were limited to the mere assessment of alcohol consumption. The frequent association of alcohol and cocaine is different from other patterns of use in North Europe countries.

Alcohol drinking, mean corpuscular volume of erythrocytes, and alcohol metabolic genotypes in drunk drivers.

Regular and irregular abuse of alcohol are global health priorities associated with diseases at multiple sites, including cancer. Mechanisms of diseases induced by alcohol are closely related to its metabolism. Among conventional markers of alcohol abuse, the mean corpuscular volume (MCV) of erythrocytes is prognostic of alcohol-related cancer and its predictivity increases when combined with functional polymorphisms of alcohol dehydrogenase (ADH1B [rs1229984] and ADH1C [rs698]) and the mitochondrial aldehyde dehydrogenase (ALDH2 [rs671]). Whether these genetic variants can influence abuse in alcohol drinking and MCV has never been examined in drunk-driving traffic offenders. We examined 149 drunk drivers, diagnosed as alcohol abusers according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth edition Text Revision (DSM-IV-TR) and enrolled in a probation program, and 257 social drinkers (controls), all Caucasian males. Alcohol intake was assessed according to self-reported drink-units/d and MCV unadjusted and adjusted for age, smoking, and body mass index. Multivariable models were used to compute MCV adjusted means. Genotype analyses were performed by PCR on DNA from blood. The adjusted MCV mean was higher in drunk-driving abusers than in controls (92 vs. 91fL; P<.0001) and increased with the number of drink-units/d in both abusers and controls (P-trend=.0316 and .0089) already at intermediate quantities (0-1 vs. 2-4 drink-units/d: P=.054 and .024). Carriers of the common ADH1B*1/*1 (rs1229984) genotype were more likely to be drunk-driving abusers (P=.008), reported higher drink-units/d (P=.0126), and had larger MCV (P=.035). The rs698 ADH1C and rs671 ALDH2 polymorphisms were not associated with MCV. ADH1B*1/*1 polymorphism is significantly associated with being a drunk-driving abuser, higher alcohol drinking, and MCV enlargement. This suggests that drunk drivers with augmented MCV modulated by the alcohol metabolic ADH1B*1/*1 genotype may be at higher risk of driving incapability and of alcohol-related cancer.

No‐discharge atmospheric pressure chemical ionization mass spectrometry of ethyl glucuronide and ethyl sulfate

Substance use disorders are a major public health problem in Western countries, and alcohol is certainly the most common substance of abuse/dependence in patients presenting for treatment. In addition, about 4% of the global burden of disease is attributable to alcohol, which accounts for about as much death and disability globally as tobacco and hypertension.[1] In this context, biological markers of ethanol consumption are becoming more and more important in both clinical and forensic settings to prove recent use and/or chronic alcohol abuse. In the human organism, 90–95% of ethyl alcohol is rapidly eliminated by oxidation, mainly in the liver via alcoholand aldehyde-dehydrogenase, catalase, and microsomal ethanol-oxidizing systems. Minor amounts are excreted by the kidneys (0.5–2%), lungs (1.6–6%), and skin (max. 0.5%).[2] The detoxifying pathway of alcohol elimination via ethyl glucuronide (EtG) and ethyl sulfate (EtS) is reported to represent 0.5–1.5% of total ethanol elimination.[3,4] Nevertheless, until recently, only limited attention has been paid to this pathway in comparison to hepatic and extra-hepatic oxidative metabolism. EtG and EtS are nonvolatile, water-soluble nonoxidative ethanol metabolites that can be quantified in body fluids and tissues for an extended time period after complete elimination of alcohol, being respectively detectable in serum up to 8 h[5] and in urine up to 80 h after ethanol elimination.[6] These metabolites close the gap in the detection window between short-term markers (e.g. ethanol) and long-term markers like carbohydrate deficient transferrin, gamma-glutamyl transpeptidase, and mean corpuscular volume, generally used to diagnose a chronic alcohol abuse. The detection of alcohol consumption that has taken place several days or weeks earlier, via the determination of EtG and EtS, represents a useful forensic tool as well as a possible method for monitoring alcohol consumption in patients being treated for alcoholism.[7] A number of analytical methods have been developed for the determination of EtG and EtS in biological matrices such as gas chromatography-mass spectrometry with derivatization,[5,8] liquid chromatography-mass spectrometry (LC-MS),[9] liquid chromatography-tandem mass spectrometry (LC-MS/MS),[10,11] liquid chromatography with pulsed electrochemical detection,[12] capillary zone electrophoresis,[13,14] or immunochemical tests.[15] The use of electrospray ionization (ESI) and LC-MS/MS methods on the most recent high-performance mass spectrometric instrumentation enabled the mise au pointe of methods fulfilling guideline requirements for forensic confirmation and exhibiting low limits of detection (LOD) and low limits of quantification (LOQ) for EtG, EtS, and ethylphosphate after simple or null sample pretreatment.[16] However, there are still cases that point out the limits of the ESI technique with respect to an efficient analyte ionization (and thus analyte detection). For instance, the analysis of hydrophilic compounds such as sugars is still not so effective using high-flow rate techniques. Furthermore, it has been described that ESI can suffer from ‘matrix effects’,[17,18] i.e. the combined effect of all components of the sample other than the target analyte on the measured analyte quantity.[19] During the phase of ion evaporation from the charged droplet and/or charged droplet fission, the matrix effect reflects on the privileged ionization of some neutral species in presence of others. Furthermore, the existence of different mechanisms[20 – 23] in the ESI may reflect on the final ion signal yield, as a result of the geometry of the ion source. In particular, on axis ESI interfaces suffer from the heavy burden of high throughput analyses and require frequent maintenance in order to restore the initial sensitivity. As a consequence, even though EtG and EtS exhibit pKa of 3.21 and 3.14[13,24] and would produce the corresponding [M−H]− anions in neutral/basic solutions, the efficiency of their ESI is indubitably related to the chemical properties of the analytes in the ESI droplets, their equilibrium partitioning between the inner and outer regions of droplets, and the presence of contaminants or other analytes that might suppress the analyte signal. In the development of an LC-MS method for the quantitative determination of EtG and EtS in a large number of urine samples, such as those originating by a driving licence regranting protocol, we initially moved towards the use of an ESI interface, as suggested by outstanding literature data.[11,16,25,26] All mass spectrometric measurements were obtained using an LCQ (Finnigan, San José, CA, USA) ion trap mass spectrometer equipped with either an ESI or an atmospheric pressure chemical ionization (APCI) source and operating in negative ion mode. EtG and d5-EtG were purchased from Medichem (Promochem, Milan, Italy); EtS and d5-EtS were prepared by chemical esterification of sulfuric acid with ethanol and d5-ethanol. Water was obtained from a Milli-Q Academic system (Millipore, Bedford, MA, USA). Acetonitrile (Sigma, Milano, Italy), formic acid (Sigma, Milano, Italy), and methanol (Fluka, Milano, Italy) were of HPLC grade. The negative ion ESI spectra of EtG and EtS, obtained by direct infusion of their 2.5 μg/ml aqueous solutions at a flow rate of 40 μl/min, with concomitant infusion of mobile phase A (water, 0.1% formic acid, 10 mmol/l ammonium formate, pH 3, flow rate of 0.2 ml/min) and acetonitrile (0.4 ml/min) are reported in Fig. 1. The optimized ESI parameters were: sprayer voltage + 4 kV, entrance capillary temperature 160 ◦C, entrance capillary voltage 10 V, sheath gas (nitrogen) 60 arbitrary units (a.u.), entrance capillary temperature 160 ◦C. The molecular anions [M−H]− of both analytes are detected at m/z 221 and 125, respectively, in a quite high intensity (>106 a.u.). In both cases, some ionic species are visible at higher m/z values; for EtG, these ions may be rationalized by a dimerization process ([2M−H]−, m/z 443) and the formation of ‘clusters’ with formic acid ([M+HCOOH−H]−, m/z 267). Interactions between the carboxylic groups have already been described for organic acids as originating in the condensed phase;[28] formation of the observed ion clusters was partially inhibited by applying an in-source de-fragmentation potential of 5 V for EtG and d5-EtG, and of 15 V for EtS and d5-EtS. The development of an LC-ESI-MS/MS method for the determination of EtG and EtS in biological fluids was then undertaken. However, when multiple LC injections of urine extracts were performed, the instrumental responses related to EtG, EtS, and the corresponding deuterated standards showed a rapid decrease and rapidly fell to zero. This phenomenon was not observed at the same, critical extent for other compounds which are routinely analyzed by LC-ESI/MS in our laboratory with the same instrument. The original instrumental sensitivity for EtG and EtS could be restored only after an accurate cleaning of the entrance capillary, or its substitution. The urinary matrix effect, that was at first invoked as a possible explanation for the ion current decrease, was ruled out on the basis of direct infusion experiments: when infusion of a standard solution of EtG, EtS, Caffeine, and Ultramark was protracted for more than 10 min, the ion current related to EtG and EtS fell to zero, whereas the signal of Caffeine and Ultramark remained constant. Considering that minute droplets originating from the ESI process are capable of inflowing the entrance capillary together with unsolvated ions, the observed phenomenon may be explained by the deposit of neutral species on the entrance capillary inner surface. This occurrence should be particularly relevant when an on axis ESI source geometry is employed, as in the present case. At high temperature these neutrals may undergo thermal decomposition and activate the inner surface against EtG and EtS. As a matter of fact, a linear, reproducible decreasing of the [M−H]− signal was observed when the entrance capillary was heated from 160 to 300 ◦C.

An asthmatic patient with progressive lung dysfunction: a case of misdiagnosis

In May, 2005, a 40-year-old installer of thermohydraulic systems, who was a heavy smoker and had asthma, was referred to our hospital for progressive lung dysfunction of mixed type (Tiff eneau index 57%; forced expiratory volume in 1 s 1·73 L, 43% predicted; total lung capacity 5·75 L, 80% predicted; vital capacity 3·26 L, 64% predicted). He had a history of an acute episode of pneumonitis with asthma exacerbation and pleural eff usion in March, 2005. High-resolution CT showed wandering bilateral nodular shadows, more obvious in the lower right apical lobe. Blood tests showed mild eosinophilia (11·7%; normal range 1·0– 6·0), increased IgE (567 kU/L; 0–91), ESR (59 mm/h; 1–12), and C-reactive protein (31·1 mg/L; 0–5·0). All other laboratory results, including immunological markers, were within normal range. From the clinical (asthmatic and rhinitic symptoms), serological (eosino philia), and CT (wandering bilateral nodular shadow) fi ndings, a working diagnosis of Churg-Strauss syndrome was made. However, because of persistent eosinophilia, worsening dyspnoea, and persistent opacities on CT, the patient underwent bronchoscopy, bronchoalveolar lavage (BAL), and transbronchial biopsy (TBB). Brown fl uid (300 mL) was collected and cytofl uorimetry showed an increased cell count (250 000/mL; normal range 120 000–190 000) with recruitment of eosinophils (5%; 0–3). No informative diagnostic features were apparent from TBB. Microbiological investigations (viral, bacterial, fungal, mycobacterial) done on the BAL specimens and blood were all negative. Parasitic infestations were investigated and excluded. A lung biopsy was then taken by surgery, and histology showed a variegated appearance characterised by necrotising granulomas, with a broncho centric distribution associated with mild eosinophilic interstitial infi ltration and foci of organising pneumonia (fi gure). Staining for microorganism detection was negative. On further questioning, the patient revealed that he had used cocaine at the time of the fi rst episode of pneumonitis. He confi ded that he sporadically inhaled cocaine. After obtaining consent from the patient, we did toxicological analyses on urine and hair by gas chromatography–mass spectrometry. Urine and the 3 cm proximal hair sample were negative, but the 3 cm distal hair sample was positive for cocaine and its metabolite benzoylecgonine, allowing documentation of cocaine intake some months earlier. The patient was strongly discouraged from using cocaine and was treated with 0·5 mg/kg daily of oral prednisone for 2 weeks with progressive tapering over the subsequent 6 weeks. He improved within a few days, and 2 months after the start of therapy showed complete clinical and radiological resolution. He was well at last follow-up in February, 2006. Abuse of illicit inhaled substances has become more common, increasing concern about potential pulmonary and other medical complications. Cocaine use can cause various pulmonary abnormalities associated with a broad range of histopathological changes. Pre-existing atopy and asthma seem to be major risk factors for exacerbation of bronchoconstriction and, in our patient, could have resulted in slow resolution of lung lesions after exposure to cocaine. Our patient developed lung dysfunction of a mixed type, which suggests structural damage not only to the bronchial wall but also to alveolar units. Clinical reports of acute pulmonary oedema and diff use alveolar haemorrhage in temporal association with cocaine use, as well as autopsy evidence of pulmonary haemorrhage and interstitial disorders, provide support for the concept of cocaine-induced damage to the alveolar-capillary membrane. Clinicians should bear in mind that a confi dential dialogue with patients is fundamental to obtain personal information about lifestyle and social attitudes so that an accurate diagnosis can be made. In our case, the appropriate diagnosis led to a dramatic change in the patient’s prognosis and avoided long-lasting high-dose corticosteroid therapy.