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A novel approach to the analysis of Carbohydrate-Deficient Transferrin (CDT) based on capillary electrophoresis with in-line immunosubtraction.
No full textLa diagnosi oggettiva di abuso di alcol ha una grande rilevanza in diversi settori della medicina clinica e forense, tra cui gastroenterologia, cardiologia, neurologia e psichiatria, medicina del lavoro e del traffico, ecc patologia forense.Dai dati della Società Italiana di Alcologia emerge che in Italia sono circa 32 milioni le persone che bevono regolarmente, mentre sono 3.5 milioni coloro che bevono in maniera inadeguata,cioè che arrivano a manifestare sintomi da intossicazione acuta. Gli alcolisti, ovvero i soggetti alcool dipendenti con patologie correlate, sono circa un milione, mentre i decessi per cause alcol-correlate sono circa 30.000 all’anno, dato significativo se paragonato ai 1000 decessi/anno per tossicodipendenza.
L’uso di alcol in Italia rappresenta la quarta causa di morte.
Negli ultimi anni, in aggiunta al tradizionale approccio diagnostico basato su enzimi epatici, MCV e gamma-GT, è stato introdotto nella pratica di laboratorio un biomarker più specifici, come la transferrina carboidrato carente (CDT).
La transferrina sierica è una molecola con peso molecolare di 79,5 KDa contenente due catene di polisaccaridi, tali catene hanno strutture ad antenna con due, tre o quattro ramificazioni e ogni antenna termina con una molecola di acido sialico. Nel siero si trovano molecole di transferrina prive di acido sialico dette asialo-Tf e altre forme con più o meno acido sialico fino ad un massimo di otto.
La CDT è un gruppo di glicoforme minori della transferrina, che comprende asialo-Tf, monosialo-Tf, diasialo-tf, la cui concentrazione sierica, come ampiamente riportato in letteratura, è strettamente correlata con l'assunzione cronica di alcol.
A causa delle importanti conseguenze personali e legali della diagnosi di abuso di alcol, è importante avere metodo specifico e selettivo per la determinazione di questo marcatore.
L’obiettivo analitico consiste nell’effettuare misure quantitative delle isoforme CDT rispetto alle isoforme non-CDT. Ma la somiglianza strutturale tra isoforme CDT e non-CDT, la bassa concentrazione delle isoforme CDT rispetto alla transferrina totale, nonché la microeterogeneità della transferrina rendono indispensabile l’uso di tecniche con elevato livello di specificità e sensibilità.
L'analisi strumentale della transferrina carboidrato carente, è più comunemente svolta in cromatografia liquida ad elevate prestazioni (HPLC) o elettroforesi capillare (CZE). Tra queste due tecniche, l’elettroforesi capillare mostra una maggiore efficienza e produttività, ma presenta una selettività minore rispetto all’HPLC, a causa di una lunghezza d'onda UV meno specifica.
Per questo motivo, il presente lavoro è stato finalizzato allo sviluppo di un metodo in elettroforesi capillare per la determinazione della CDT nel siero, al fine di ottenere un’identificazione inequivocabile delle isoforme della CDT.
Le condizioni analitiche sono le seguenti: capillare uncoated , 30 m X 60 cm (L= 50 cm effettivi al detector); buffer di corsa composto da 100 mM acido borico e 6 mM DAB (1,4-Diamminobutano), pH a 8.3 con NaOH; potenziale 30 kV; temperatura 25°C; lunghezza d'onda 200 nm; secondo queste condizioni elettroforetiche, si è ottenuta una buona separazione delle isoforme della transferrina, permettendo una identificazione univoca dei picchi della CDT. Al fine di eliminare possibili interferenti e per ottenere una più specifica e chiara identificazione delle isoforme, è stata effettuata una immunosottrazione in-line, iniettando direttamente una plug di anticorpo specifico per la transferrina subito dopo il campione. Questo approccio analitico è stato quindi applicato con successo in campioni dubbi o che presentavano picchi interferenti.The objective diagnosis of alcohol abuse has a high relevance in different areas of clinical and forensic medicine, including gastroenterology, cardiology, neurology and psychiatry, occupational and traffic medicine, forensic pathology etc.
In the recent years, in addition to the traditional diagnostic approach based on liver enzymes, MCV and gamma-GT, a more specific biomarker, such as Carbohydrate Deficient Transferrin (CDT), has been introduced in the laboratory practice and is now widely used worldwide.
Transferrin glycoforms, collectively referred as CDT, include the asialo-Tf, monosialo-Tf and disialo-Tf isoforms. Moreover, CDT serum concentration is lower than 1,5% of total transferrin amount.
That an increase of the “total CDT” (asialo-Tf + monosialo-Tf + disialo-Tf) occurs after a persistent alcohol intake was confirmed in the literature.
The instrumental analysis of Carbohydrate Deficient Transferrin (CDT), is most commonly carried out by High Performance Liquid Chromatography (HPLC) or Capillary Zone Electrophoresis (CZE). Between these two techniques, CZE shows higher efficiency and productivity, but is often reported to be inferior to HPLC in terms of selectivity, because of a less specific UV detection wavelength than HPLC. On these grounds, the present work was aimed at the development of an improved CZE method for CDT determination including an on-line immunosubtraction step specifically aimed at enhancing the analytical specificity of CZE determination. The analytical conditions were as follows: uncoated fused silica capillary, 30 m X 60 cm (L= 50 cm to detector); running buffer, 100 mM borate and 6 mM DAB (1,4-Diaminobutane), pH 8.3; voltage, 30 kV; temperature, 25°C; detection, 200 nm. Under the described CZE conditions, a baseline separation between all the CDT related peaks was achieved with good analytical performances in terms of both precision and accuracy. In order to achieve unequivocal recognition of the CDT peaks, an in-capillary immunosubtraction step was included by loading a plug of anti-human transferrin antibody solution after the sample plug. This analytical approach was applied successfully to recognize CDT peaks in the presence of potential interferences
Rapid determination of lithium in serum samples by capillary electrophoresis
No full textLithium salts are still one of the most popular therapeutic approaches to the treatment of bipolar disorders, notwithstanding the introduction of more modern, less toxic drugs. Because of a narrow therapeutic range, lithium serum concentrations must be strictly monitored during the treatment to avoid life-threatening neurotoxicity. For this purpose, methods based on flame photometry or ion-selective electrodes are usually applied. The aim of the present work was to develop and validate a simple method for the determination of lithium in serum based on capillary zone electrophoresis with indirect detection. A validation of the method was carried out, including a comparison with an automated routine method based on ion-selective electrode
Determinazione di Metadone ed EDDP in resti scheletrici mediante LC-QQQ-MS
No full textSi presenta il caso della determinazione di metadone ed EDDP mediante metodica LC-QQQ-MS nel midollo osseo estratto da resti scheletrici, successivamente identificati come appartenuti ain vita a soggetto tossicodipendente in terapia sostitutiva con metadone. A nostra conoscenza, risulta essere la prima volta che tale sostanza è identificata in questa particolare matrice biologica
Metodo analitico per l'identificazione di almeno una glicoforma della proteina transferrina
No full textL'invenzione riguarda un metodo analitico innovativo che sfrutta una derivatizzazione specifica con terre rare della transferrina per la determinazione ad alta sensibilità della Carbohydrate Deficient Transferrin (CDT) il biomarker più affidabile delle patologie della glicosilazione proteica e dell'abuso cronico di alco
Analytical method for the identification of at least one glycoform of the transferrin protein
No full textThe patent concerns a new analytical method for the analysis of Carbohydrate Deficient Transefrrin isoforms based on lanthanide transferrin derivatization, HPLC separation and fluorescence detection
Calculation of LogP based on migration data in MEKC: application to the new synthetic cannabinoids.
No full textSince 2004, a number of “herbal blends” containing synthetic cannabinoid analogues have
appeared in the market, as a form of “legal” alternatives to Cannabis. These products are
available online through the “e-commerce” and in “smart shops” under several forms and various
brand names. A particular warning related to the diffusion of synthetic cannabinoids is based
on one hand on their high toxicological potential, and on the other hand on the insensitivity of
the current screening tests for cannabis towards these molecules. Moreover, because of their
recent introduction, the literature regarding these compounds is still limited. In particular any
experimental information on their octanol/water partition coefficient (logP) values is still lacking.
Indeed, hydrophobicity value is an important parameter to investigate drug absorption,
bioavailability and metabolism of molecules, as well as their toxicity. As a measure of molecular
hydrophobicity, the logarithm of the partition coefficient between 1-octanol and water (logP) is
widely used. The aim of the present study was to calculate the LogP of synthetic cannabinoids
by using micellar electrokinetic chromatography (MEKC) with UV detection. Samples were
analyzed using a fused silica capillary (30 mm x 40 cm) and a 25 mM sodium borate buffer pH
8, containing30 mMSDS and n-propanol 20%. After having constructed a calibration line using
10 appropriate standards with known LogP, an EOF marker and a micelle marker, a good
correlation was found between the MEKC retention data of further 5 compounds with known
LogP. The same calibration line was used to calculate the unknown LogP of the new synthetic
cannabinoids, namely JWH-200, AM-694, JWH-250, JWH-073, JWH-015, JWH-018, JWH-081,
JWH-122, JWH-019, JWH-210. The resulting Log P were in the range from 2.9 to 5.15. An
acceptable agreement was found between the experimental data and a few LogP values
calculated with XLogP3-AA reported in the literature
Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation
No full textThis 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
Analysis of drugs of forensic interest with Capillary Zone Electrophoresis / Time-of-Flight Mass Spectrometry based on the use of non-volatile buffers
No full textThe present work is aimed at investigating the influence of the background electrolyte composition and concentration on the separation efficiency and resolution and mass spectrometric detection of illicit drugs in a CZE-ESI-TOF MS system. The effect of phosphate, borate and Tris buffers on the separation and mass spectrometry response of a mixture of 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, methadone, cocaine, morphine, codeine and 6-monoacethylmorphine was studied, in comparison with a reference ammonium formate separation buffer. Inorganic non volatile borate and Tris buffers proved hardly suitable for CE-MS analysis, but quite unexpectedly ammonium phosphate buffers showed good separation and ionization performances for all the analytes tested. Applications of this method to real samples of hair from drug addicts are also provided
Metal Interactions in the Ni Hyperaccumulating Population of Noccaea caerulescens Monte Prinzera
Full text linkHyperaccumulation is a fascinating trait displayed by a few plant species able to accumulate large amounts of metal ions in above-ground tissues without symptoms of toxicity. Noccaea caerulescens is a recognized model system to study metal hyperaccumulation and hypertolerance. A N. caerulescens population naturally growing on a serpentine soil in the Italian Apennine Mountains, Monte Prinzera, was chosen for the study here reported. Plants were grown hydroponically and treated with different metals, in excess or limiting concentrations. Accumulated metals were quantified in shoots and roots by means of ICP-MS. By real-time PCR analysis, the expression of metal transporters and Fe deficiency-regulated genes was compared in the shoots and roots of treated plants. N. caerulescens Monte Prinzera confirmed its ability to hypertolerate and hyperaccumulate Ni but not Zn. Moreover, excess Ni does not induce Fe deficiency as in Ni-sensitive species and instead competes with Fe translocation rather than its uptake
Innovation in the analysis of carbohydrate deficient transferrin (CDT) with capillary electrophoresis
No full textCDT is a widely recognized marker of chronic abuse of alcohol, ued in both clinical and forensic settings. CDT identifies a group of minor glycoforms of transferrin, the major iron transporting serum protein. Sound research has indicated that subjects with an alcohol intake > 60-80 grams per days for at least 7 consecutive days show increased CDT concentrazion. At present, the analytical methods for CDT determination include immunoassays and methods based on capillary electrophoresis and HPLC. In order to achieved unequivocal recognition of CDT peaks, an automated and reproducible in-capillary immunosubtraction was developed by injectin a plug of serum sample followed by a plug of anti-Tf antiserum
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