Evaluation of the illegal use of clenbuterol in Portuguese cattle farms from drinking water, urine, hair and feed samples.

The recent discovery of clenbuterol contamination in Portuguese food led to the specific inspection of 16 cattle farms for beta-agonists, involving the analysis of a total of 486 samples (78 feed, 106 drinking water, 168 urine and 134 hair). The samples were screened for the beta-agonists: bromobuterol, cimaterol, clenbuterol, clenpenterol, clenproperol, hydroxymethylclenbuterol, mapenterol, salbutamol and terbutaline. Only clenbuterol was found in all analyzed matrices and the most likely method of illegal administration to animals was through drinking water. Of all samples analysed, 14.15% of drinking water were found positive in the range 0.03-3.80 mg l(-1) clenbuterol. Inclusion of hair samples in the Portuguese plan for clenbuterol residue control in live animals is discussed.

Levels of ochratoxin A in serum from urban and rural Portuguese populations and estimation of exposure degree.

Urban and rural population exposure to ochratoxin A (OTA) in central zone of Portugal was investigated in three places: Coimbra, Verride and Ereira. The analytical method proposed for the determination of ochratoxin A involved extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column (IAC), high performance liquid chromatography (HPLC) with spectrofluorimetric detection (FD) for separation and identification of ochratoxin A, and confirmation with HPLC-FD after OTA methylation in serum. The limit of quantification of the proposed method was 0.1 microg/L for serum and 0.05 microg/L for blood. OTA recoveries in serum ranged from 70.3% to 115.3% for levels at 0.25 microg/L and 0.5 microg/L, respectively, with a within-day RSD between 8.0% and 16.2%. Ochratoxin A serum levels were evaluated in an hundred and four donors from Coimbra city, Verride, and Ereira. The study revealed a frequency of detection of 100%. The ratio of ochratoxin A level in serum to whole blood was 2.0+/-0.7. The overall concentrations range from 0.25 to 2.49 microg/L, 0.14 to 1.91 microg/L, and 0.19 to 0.96 microg/L, for samples of Verride, Ereira, and Coimbra, respectively. The mean concentration and standard deviation were 0.78+/-0.53 microg/L, 0.44+/-0.31 microg/L, and 0.42+/-0.18 microg/L for the same samples. A significant difference was found in Verride population (P-value=0.000). Levels of OTA are clearly higher in males from rural areas than in females. For all samples, a significant difference was found in Verride male population (P-value=0.014).

Determination of fumonisins B1 and B2 in Portuguese maize and maize-based samples by HPLC with fluorescence detection.

Fumonisins B(1) (FB1) and fumonisin B2 (FB2) are the main members of a family of mycotoxins produced by Fusarium verticillioides, Fusarium proliferatum, and other fungi species of the section Liseola. The present work shows the results of comparative studies using two different procedures for the analysis of fumonisins in maize and maize-based samples. The studied analytical methods involve extraction with methanol/water, dilution with PBS, and clean-up through immunoaffinity columns. Two reagents (o-phthaldialdehyde and naphthalene-2,3-dicarboxaldehyde) were studied for formation of fluorescent derivatives. The separation and identification were carried out by high-performance liquid chromatography with fluorescence detection. The optimized method for analysis of fumonisins in maize involved extraction with methanol/water (80:20), clean-up with an immunoaffinity column, and derivatization with naphthalene-2,3-dicarboxaldehyde (NDA). The limit of detection was 20 microg kg(-1) for FB1 and 15 microg kg(-1) for FB2. Recoveries of FB1 and FB2 ranged from 79% to 99.6% for maize fortified at 150 microg kg(-1) and 200 microg kg(-1), respectively, with within-day RSDs of 3.0 and 2.7%. The proposed method was applied to 31 samples, and the presence of fumonisins was found in 14 samples at concentrations ranging from 113 to 2,026 microg kg(-1). The estimated daily intake of fumonisins was 0.14 microg kg(-1) body weight per day.

A comparative study of extraction apparatus in HPLC analysis of ochratoxin A in muscle.

Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus and by Penicillium verrucosum, that occurs in meat products. The aim of this work was to optimize an efficient extraction procedure for the determination of OTA in muscle tissue in order to assess its occurrence in meat samples. Three different apparatus, a Waring blender, a switching apparatus, and an ultrasonic processor, were evaluated to verify the efficiency of extraction. The analytical methods proposed involve the extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column, high-performance liquid chromatography/fluorescence detection for separation and identification of OTA, and confirmation with liquid chromatography/FD after methylation of OTA in muscle tissue. The limit of quantification of the proposed method was 0.04 microg kg(-1). Recoveries of OTA, using switching apparatus, ranged from 90.3 to 103.2% for chicken muscle spiked at 2.4 and 0.48 microg kg(-1), respectively, with a within-day relative standard deviation of 17 and 15.3%. The proposed method was applied to 38 chicken, swine, and turkey muscle samples and the presence of OTA was confirmed in five samples. Finally, the estimated daily intake of OTA in this study was between 23 pg kg(-1) body weight per day for swine samples and 18 pg kg(-1) body weight per day for turkey samples.

Organochlorine pesticide residues in European sardine, horse mackerel and Atlantic mackerel from Portugal.

This paper reports the results for the surveillance of nine organochlorine pesticides (HCH isomers (alpha, beta, e, gamma), p,p'-DDD, p,p'-DDT, p,p'-DDE, p,p'-DDD, HCB and aldrin) in muscle of three fish species, European pilchard (Sardina pilchardus), Atlantic horse mackerel (Trachurus trachurus) and Atlantic mackerel (Scomber scombrus). Analytical methodology included n-hexane extraction, clean-up with 2% deactivated Florisil, and quantification with gas chromatography-electron capture detection (GC-ECD). The highest mean concentrations were found for p,p'-DDT in sardine and mackerel at levels of 30.1 and 109.9 microg kg(-1), respectively, and for p,p'-DDD in horse mackerel at 51.9 microg kg(-1). Three species had higher levels for S-DDT than S-HCH. The estimated daily intake of organochlorine pesticides in the three species showed that in sardine, the highest EDIs were found for aldrin, at 1.8 ng kg(-1) bw day(-1), which represents 1.8% of the acceptable daily intake (ADI), and for ss-HCH, at 4.0 ng kg(-1) bw day(-1), representing 0.4% of ADI. Lowest values were found for Atlantic mackerel. Statistical analysis to determine the differences in mean concentrations of pesticides between species, and any correlation between groups of residues related with each one of the species, was undertaken.

Validation of an analytical methodology for determination of oxytetracycline and tetracycline residues in honey by HPLC with fluorescence detection.

An analytical method for the determination of OTC and TC residues in honey was developed. Sample treatment involves an extraction in EDTA-McIlvaine buffer, followed by a solid-phase cleanup step. With regard to the cleanup procedure, different SPE cartridges were evaluated and the results presented. The method was validated according to the guidelines laid down by the 2002/657/EC European Decision parameters: decision limit (Cc alpha) and detection capability (CC beta) were 20 and 21 microg/Kg and 49 and 50 microg/Kg for OTC and TC, respectively, and recoveries of OTC and TC from spiked samples, at three fortification levels, were higher than 87% for both compounds. The analytical method was applied to 57 honey samples.

Survey of caffeine levels in retail beverages in Portugal.

The caffeine content of 85 retail beverage samples purchased from local supermarkets between 1995 and 2004 was determined. The potential intake of caffeine through the consumption of these beverages (but excluding coffee) was estimated for students of the University of Coimbra, Portugal. The caffeine content of the beverages ranged from 47.5 to 282.5 mg l(-1) for teas, from 20.1 to 47.2 mg l(-1) for tea extracts samples, and from 80.7 to 168.7 mg l(-1) for cola soft drinks. Caffeine was not completely absent from caffeine-free colas, and energy drinks had a far greater caffeine content than regular drinks, ranging from 21 to 2175 mg l(-1). Soft drinks were consumed by 72% of the individuals, although 14% of the survey participants did not drink any of the different types of the beverages studied. Contrary to expectations for this age group, no consumptions of energy drinks was reported. Daily caffeine intake was estimated to range from 4.7 to 200 mg day(-1), but with only 5% reporting a daily intake around 200 mg caffeine. Cola-type beverages were an important dietary source of caffeine for the population studied. Statistical differences in the caffeine intake between the male and female populations were found, with p = 0.014, being higher for the male population. Of the beverages studied, cola-type drinks showed statistical differences for the male population, p = 0.03, and tea showed statistical differences for female population p = 0.013, respectively.

Determination of organochlorine pesticide residues in honey from the central zone of Portugal and the Valencian community of Spain.

In this study nine organochlorine pesticide residues (alpha-, beta-, and gamma-hexachlorocyclohexane (HCH), hexachlorobenzene (HCB), aldrin, p,p'-DDE, p,p'-DDD, o,p'-DDT, and p,p'-DDT) in forty nine samples of honey collected from markets of Portugal and Spain during 2001 and 2002, respectively, were evaluated. For this evaluation, three analytical procedures were studied. The analytical procedure, based on LLE extraction with ethyl acetate followed by gas chromatography-electron-capture detection (GC-ECD) for quantification, and mass spectrometry (GC-MS) for confirmation, has been selected. Recoveries of spiked samples ranged from 68%, for beta-HCH, and 126% for p,p'-DDT, for fortification levels between 10 and 100 microg/kg, and 64%, for alpha-HCH, and 143% for gamma-HCH for fortification levels between 20 and 200 microg/kg. Limits of quantification, using GC-ECD, were from 0.01 and 0.10mg/kg, and limits of detection between 0.001 and 0.02 mg/kg. Fourteen Valencian samples were contaminated, containing residues of HCB or/and HCH isomers. The frequency of detection was 56% for Spanish samples. In Portugal, 23 samples were contaminated, what means 95.8%. In Spanish samples, concentrations range from nd to 0.03 mg/kg for HCB, and nd to 2.24 mg/kg for HCH-total. The mean concentration and standard deviation were 0.017+/-0.011 mg/kg for HCB, and 0.579+/-0.747 mg/kg for HCH-total, contributing the gamma isomer with the highest values. The samples from Portugal showed higher levels. Levels of HCB ranged from nd to 0.39 mg/kg. HCH-total ranged from nd to 4.86 mg/kg, and DDT-total from nd to 0.658 mg/kg. Mean concentration and standard deviation were 0.09+/-0.116 mg/kg for HCB, 1.357+/-1.30 mg/kg for HCH-total, and 0.143+/-0.193 mg/kg for DDT-total.

alpha-Tocopherol modulates liver toxicity of the pyrethroid cypermethrin.

The objective of the current study was to analyze the hepatotoxic effect caused by cypermethrin (CYP) in rats, and to evaluate the possible protective effect of the antioxidant alpha-tocopherol (alpha-T). Fifty male Wistar rats were given daily i.p. doses of 300 mg/kg per day of CYP during 7 days. Half of them were administered three previous doses of 100 mg/kg per day of alpha-T, followed by seven subsequent oral doses of 40 mg/kg per day of alpha-T. The levels of biochemical indicators and histological liver damage were determined, as well as DCVA in urine. CYP altered the lipid metabolism. Such alterations were inhibited 32% by alpha-T, except for LDL. Alterations in AST were modulated in 29%. In the histology, alpha-T reduced mitochondria damage, and swelling of the endoplasmic reticulum of the liver cells. The results suggest that alpha-T can modify CYP metabolism, changing the lipidic profile and the histological analysis.

Changes in stiffness induced by hindlimb suspension in rat Achilles tendon.

The aim of this study was to measure the effects of hindlimb suspension on mechanical properties of the rat Achilles tendon. Adult male Wistar rats were randomly assigned to groups to be either suspended, or a control. After 21 days, Achilles tendons were removed for mechanical analysis. Classical tests of tensile performance were made, and mechanical parameters were derived from a stress-strain relationship. The tendons of animals that had been suspended presented values for maximal stress and tangent modulus which were 37.5% (P < 0.01) and 41% (P < 0.01), respectively, lower than the tendons of the control rats. In a similar way, the energy absorption capacity had largely decreased in animals that had been suspended. However, the maximal strain was similar in the two groups. These results showed that hindlimb suspension in rats has an important detrimental effect on mechanical properties of the Achilles tendon. Differences in tendon stiffness obtained here, along with those found by other investigators, encourage the hypothesis that homeostatic responses of soft tissues are due to changes in limb loadings. This study may be useful in providing a better understanding of the adaptation of human skeletal muscle when exposed to microgravity.

Determination of organochlorine pesticide residues in medicinal plants sold in Coimbra, Portugal.

Levels of 14 organochlorine pesticide residues--1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) group (p,p'-DDT, o,p'-DDT, p,p'-DDD, and p,p'-DDE), HCH isomers (alpha-, beta-, and gamma-HCH), hexachlorobenzene (HCB), aldrin, dieldrin, endrin, heptachlor, alpha-endosulfan, and endosulfan sulfate--were evaluated in 127 samples of medicinal plants collected in pharmacies (78 samples) and herb stores (49 samples) in 1996. Samples were divided between 15 national brands and 7 foreign brands. Most samples sold in pharmacies contained residues of gamma-HCH (51.3%). All residues were detected in analyzed samples, with exception of endrin in herb store samples. Detection frequency varied between 51.3% for gamma-HCH and 1.3% for endrin in pharmacy samples, and between 34.7% for HCB and 4.1% for endosulfan sulfate in herb store samples. Maximum residue levels were exceeded in 38 (48.7%) pharmacy samples and in 26 (53.1%) medicinal herb store samples.

Reflex and muscular adaptations in rat soleus muscle after hindlimb suspension.

Reflex, mechanical and histochemical adaptations of the soleus muscle following 3 weeks of hindlimb suspension (HS) were measured in the rat. HS transformed the soleus muscle fibre type composition from predominantly slow, type I, to approximately equal proportions of fast, type II and slow fibres. Consistent with this transformation was an increase in the maximum shortening velocity, V(max), and a decrease in the stiffness of the series elastic component. Disuse also produced muscle atrophy and a resultant decrease in twitch and tetanic force. Reflex responses of the ankle extensors were also obtained at 5 and 9 weeks of age for six control rats (C group) and six rats subjected to HS for 3 weeks (HS group). The soleus reflexes to a mechanical tap applied to the Achilles tendon (T reflex) and to an electrical stimulation of the sciatic nerve (H reflex) were measured. The maximal amplitude of these reflexes (T(max) and H(max)) were normalised to the maximal direct motor response (M(max)) and the T(max)/H(max) ratio was also calculated to give an index of the relative adaptations of the peripheral and central components of the reflex pathway. The HS group showed significantly higher H reflex gains than the C group, possibly due to changes in synaptic efficiency after HS. Conversely, the HS group presented strongly inhibited T reflexes and negative gains for the T(max)/H(max) ratios. This result indicated a reduced spindle solicitation after HS, which may reflect changes in the spindle sensitivity itself, but it could also be due to the decrease in stiffness of the musculo-tendinous elements in series with the muscle spindles. Such mechanical changes may play an important part in the decreased T reflex responses.

Methods for the determination of organochlorine pesticide residues in human serum.

The effectiveness of solid-phase extraction with Florisil for the determination of 12 organochlorine pesticide residues from human serum was examined. Recoveries greater than 84% and coefficients of variation better than 19% were obtained. Others methods, such as column partition and matrix solid-phase dispersion, were compared. The better method provides quantification limits ranging from 1.08 microg/l for gamma-HCH and 37.5 microg/l for p,p'-DDT when capillary gas-liquid chromatography with electron-capture detection is used for the final determination.

Beta2-adrenergic agonist residues: simultaneous methyl- and butylboronic derivatization for confirmatory analysis by gas chromatography-mass spectrometry.

A derivatization procedure for confirmatory residue analysis of beta2-agonists is described. Methyl (MBA) and butyl (BBA) boronic acids are simultaneously used for the derivatization of tulobuterol, mabuterol, mapenterol, salbutamol, clenproperol, clenbuterol, clenpenterol and bromobuterol by GC-MS determination. A temperature of 55 degrees C during 60 min was selected as optimal temperature-time condition for simultaneous MBA and BBA beta2-agonists derivatization. It was also observed that stability of boronic derivatives was maintained at -20 degrees C over a period of four days. The proposed methodology was tested in urine and it could be applied for confirmatory residue analysis of clenbuterol-like compounds.

Fungemia in cancer patients in Brazil: predominance of non-albicans species.

The objective of this study was to characterize the epidemiology of candidemia in cancer patients in the city of Rio de Janeiro, Brazil. An 18-month survey of fungemia in patients with cancer was undertaken in three Hospitals in Rio de Janeiro. Forty-three episodes of candidemia were identified in 43 patients, 43 of which were episodes of candidemia; in ten case the strains were not available for further identification of species and were excluded from this analysis. The overall distribution of fungi causing fungemia was: Candida albicans (5), Candida tropicalis (16), Candida parapsilosis (6), Candida guilliermondii (4), Candida lusitaniae (1) and Candida stellatoidea (1). Antifungal prophylaxis had been administered before the episode of fungemia in only six patients (18.2%): oral itraconazole in three patients and oral nistatin, low dose intravenous amphotericin B and oral fluconazole in one patient each. There was no difference in the presence of risk factors, clinical characteristics or in the outcome between albicans and non-albicans species, nor between Candida tropicalis and other non-albicans species. There was a clear predominance of non-albicans species, regardless of the underlying disease, antifungal prophylaxis or the presence of neutropenia.

Risk factors for death among cancer patients with fungemia.

In order to identify prognostic factors for death among cancer patients with fungemia, an 18-month survey of fungemia in patients with cancer was undertaken in three hospitals in Rio de Janeiro. For the assessment of risk factors for death, the following variables were analyzed: age; gender; underlying cancer; last treatment for the underlying disease; previous surgery; use of antibiotics, antifungal agents, steroids, or total parenteral nutrition; use of a central venous catheter; chemotherapy; radiotherapy; presence and duration of neutropenia; etiologic agent of the fungemia; treatment of the fungemia; clinical manifestations; and performance status (Karnofsky score) on the day of the positive blood culture. In multivariate analysis, the variables associated with an increased risk for death were older age, persistent neutropenia, and low performance status. Identifying risk factors for death may help to define a group-risk patients for whom new therapeutic options should be tried.

Occurrence of beta 2-adrenergic agonist residues in urine of animal meat producers in Portugal.

Between January 1, 1991, and December 31, 1993, bovine urine was collected for analysis of beta 2-adrenergic agonist residues. A multiresidue method was developed that was capable of detecting clenbuterol, clenpenterol, clenproperol, mabuterol, mapenterol, bromobuterol, tulobuterol, and salbutamol through enzyme-linked immunosorbent assay, liquid chromatography (LC) with photodiode array detection, and gas chromatography with mass detection in electron impact mode with selective-ion monitoring. Among 1031 samples, 24 contained clenbuterol and 1 contained salbutamol. The results confirm that clenbuterol is the beta 2-adrenergic agonist most used as bovine growth promoter in Portugal.

Influence of plyometric training on the mechanical impedance of the human ankle joint.

The objective of this work was to study the effects of plyometric training on the mechanical properties of the ankle joint in humans. Changes in the mechanical parameters of this musculo-articular structure were quantified with the aid of a sinusoidal perturbation technique. This technique allowed the expression of the mechanical impedance of the musculo-articular system in terms of stiffness, viscosity and inertia. Measurements were performed under passive conditions and when the subject performed plantar flexion. A 7-week period of training induced a decrease in the slope of the relationship between stiffness and plantar flexion torque, whereas passive stiffness was increased. A slight decrease in viscosity and an invariability in inertia were also found. These results are interpreted in terms of the possible adaptations of the musculo-articular structure and ultrastructure involved in the performance of plantar flexion.

Neuromuscular adaptations in rats trained by muscle stretch-shortening.

The aim of this study was the analysis of neurophysiological, mechanical and histochemical parameters to demonstrate muscle adaptation with training. If the parameters studied were to show correlated changes, it would be possible to propose that the neural and the muscle components of motor units are both affected by the training programme used. The training consisted of repeated stretch-shortening cycles known to use extensively fast fibres. After the training period electromyographical reflex activities of the ankle plantar-flexors were recorded in awake rats and then mechanical and histochemical measurements were made on isolated soleus muscles of the control and trained rats. The reflexes studied were the H-response to electrical stimulation of the sciatic nerve and the T-response to an Achilles tendon tap. The H-response analysis indicated a decrease in reflex excitability of the trained muscles. The trained soleus muscle also presented a higher contractility as demonstrated by significantly smaller twitch contraction times and higher maximal velocities of shortening measured during tetanic contractions. The reflex and contractile muscle changes were accompanied by relative increases in the number of type II fibres. The T-response was not significantly modified by training despite the decrease in motoneuron excitability demonstrated by the decrease in H-response. This would suggest that the peripheral components of the reflex pathway such as tendon stiffness and/or spindle sensitivity might be modified by training. This would imply that both the motor and the sensory parts of a muscle are affected by training.

Effects of stretch-shortening cycle training on mechanical properties and fibre type transition in the rat soleus muscle.

The effects of exercise training on mechanical properties and fibre type transitions have been investigated in rat soleus muscles. The exercise was a repetition of stretch-shortening cycles. A method of dual controlled releases was applied to obtain tension/extension curves, which characterize the elastic behaviour of the series elastic component (SEC), and the force/velocity relationship characterizing the contractile elements. Other contractile measurements included: contraction time (tc), half-relaxation time (t1/2) and twitch/tetanus ratio (Pt/Po). Additionally, the muscle fibre type composition was determined by a classical histochemical method. A 12-week period of training induced a significantly higher percentage of fast-twitch fibres and a lower percentage of slow-twitch fibres (P < 0.01). This fibre adaptation led to a significant (P < 0.01) decrease in tc and an increase in maximum shortening velocity (Vmax). An increase in compliance of the SEC was also observed. This elastic adaptation is interpreted in terms of modification of the active components of the SEC. All the histochemical and mechanical data presented in this study show that rat soleus muscles trained by stretch-shortening cycles acquired faster characteristics. Thus the results confirm that a slow-twitch to a fast-twitch fibre transition is obtainable in mature rats.