Urinary di-(2-ethylhexyl)phthalate metabolites in athletes as screening measure for illicit blood doping: a comparison study with patients receiving blood transfusion.

BACKGROUND: Subjects submitted to intravenous (IV) blood transfusions for medical reasons or blood doping to increase athletic performance are potentially exposed to the plasticizer di-(2-ethylhexyl)phthalate (DEHP) found in IV bags. Exposure to DEHP has been evaluated by measuring DEHP metabolites in selected groups of subjects. STUDY DESIGN AND METHODS: Urinary DEHP metabolites, mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), and mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP) were measured in a control group with no explicit known exposure to DEHP (n = 30), hospitalized patients receiving blood transfusions (n = 25), nontransfused hospitalized patients receiving other medical care involving plastic materials (n = 39), and athletes (n = 127). Patients were tested in the periods 0 to 24 and 24 to 48 hours after exposition. RESULTS: Urinary concentrations of all three DEHP metabolites were significantly higher in patients receiving blood transfusion than in nontransfused patients and the control group, except for MEHHP and MEOHP in the period 24 to 48 hours. Samples from four athletes showed increased concentrations of DEHP metabolites comparable to urinary concentrations of patients receiving blood transfusion. CONCLUSION: Elevated concentrations of urinary DEHP metabolites represent increased exposure to DEHP. High concentrations of DEHP metabolites present in urine collected from athletes may suggest illegal blood transfusion and can be used as a qualitative screening measure for blood doping.

Evaluation of protein N-glycosylation in 2-DE: Erythropoietin as a study case.

The structure, function, and physico-chemical properties of many proteins are determined by PTM, being glycosylation the most complex. This study describes how a combination of typical proteomics methods (2-DE) combines with glycomics strategies (HPLC, MALDI-TOF-MS, exoglycosidases sequencing) to yield comprehensive data about single spot-microheterogeneity, providing meaningful information for the detection of disease markers, pharmaceutical industry, antidoping control, etc. Recombinant erythropoietin and its hyperglycosylated analogue darbepoetin-alpha were chosen as showcases because of their relevance in these fields and the analytical challenge they represent. The combined approach yielded good results in terms of sample complexity (mixture glycoforms), reproducibility, sensitivity ( approximately 25 pmoles of glycoprotein/spot), and identification of the underlying protein. Heterogeneity was present in all spots but with a clear tendency; spots proximal to the anode contained the highest amount of tetra-antennary tetra-sialylated glycans, whereas the opposite occurred for spots proximal to the cathode with the majority of the structures being undersialylated. Spot microheterogeneity proved a consequence of the multiple glycosylation sites as they contributed directly to the number of possibilities to account for a discrete charge in a single spot. The interest of this combined glycoproteomics method resides in the efficiency for detecting and quantifying subtle dissimilarities originated from altered ratios of identical glycans including N-acetyl-lactosamine repeats, acetylation, or antigenic epitopes, that do not significantly contribute to the electrophoretic mobility, but affect the glycan microheterogeneity and the potential underlying related functionality.

Assessing the instability of the isoelectric focusing patterns of erythropoietin in urine.

IEF can be used to differentiate human urinary erythropoietin (uEPO), recombinant human erythropoietin or epoetin (rEPO) and darbepoetin (novel erythropoiesis stimulating protein (NESP)). This is the basis of the method currently used to detect misuse of rEPO and NESP by elite athletes. Recently, an unknown activity has been attributed to some urine samples (denominated 'unstable' urine by the World Anti-Doping Agency; WADA). This activity has shown to give rise to artefactual profiles for both rEPO and NESP when incubated with such urine and, thus, raised concerns with respect to doping control. We have evaluated which charges produce the characteristic IEF profiles of uEPO, rEPO and NESP and how these profiles respond to distinct enzymatic reactions. From sialidase digestions it became evident that only uEPO contains charges different from sialic acid, and a comparison of all substances after complete de-N-glycosylation localized these charges in the carbohydrate moiety. Partial desialylation, or digestion with arylsulfatase from Helix pomatia yielded profiles for recombinants species similar to those observed for unstable urine samples. The contributions from our studies to the anti-doping problem include: (i) protocols that may corroborate the potential misuse of rEPO or NESP based on the particular enzymatic activity of an arylsulfatase preparation, or a broad-specificity sialidase; (ii) assurance that the instability observed in some urine samples may only result from false-negatives, but not from false-positive testing; and (iii) a simple remedy to prevent an unstable urine from altering the IEF profile by adding selective competitive substrates.

Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine.

Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 microM) in the presence of 0.1 mM H2O2 demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60 cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis.

Dual morphometrical changes of the deltoid muscle in patients with COPD.

The present study was specifically aimed at evaluating if the structure of the deltoid muscles is modified in patients with chronic obstructive pulmonary disease (COPD). Twenty-eight male volunteers (61+/-13 yr) were assigned, according to pulmonary function, to either the COPD (n=14, FEV(1)=22-74%pred) or control group (n=14, FEV(1)=83-121%pred). Biopsies from non-dominant deltoid muscle were obtained and processed for morphometric analysis of the fibre types. Both type I and type II muscle fibres were distributed in the typical mosaic pattern. The mean value of the fibre size was within the normal range. However, three differentiated modes were observed in the deltoid from COPD patients: a central mode of normal sized fibres, a mode of atrophic fibres and a mode of hypertrophic fibres. This observation was evident even within single fascicles and especially prevalent in the most severe COPD patients. We conclude that factors with opposite effect (promotion of either atrophy or hypertrophy) exert relevant roles in the histomorphometrical characteristics of the deltoid muscles in COPD patients.