Feeding cows with hay, silage, or fresh herbage on pasture or indoors affects sensory properties and chemical composition of milk and cheese.

In European countries, silage-free feeding is an ancient tradition and has a particularly positive reputation among consumers. In the present study, we compared grass-based forages from the same plot conserved as hay or silage or fed fresh either on pasture or indoors, and we evaluated the differences in sensory properties of milk and uncooked pressed cheese. All herbage from the first cut of a grassland dominated by perennial ryegrass was harvested on the same day and preserved either as hay or silage. The first regrowth of the same plot was used for strip grazing or green feeding indoors. Balanced by breed, 24 Montbéliarde and 24 Holstein cows were allocated to the 4 treatments. Apart from the forages, the late-lactation cows received 3 kg/d of dry matter from concentrate. After 2 wk of dietary adaptation, the bulk milk of 3 subgroups, each with 4 cows, was collected. Part of the milk was pasteurized, and part was left raw and partly transformed to small-sized Cantal-type cheese ripened for 9 wk. Milk and cheese underwent descriptive sensory analysis by a trained sensory panel, as well as analyses of physicochemical traits. Volatile organic compounds of the cheeses were also analyzed. Raw and pasteurized milk from hay-fed cows had less intense odors of cooked milk, cream, and barnyard than milk from grazing cows, whereby the effect of pasteurization did not differ between herbage utilization methods. Cheeses obtained from cows fed fresh herbage (grazing and indoors) were clearly yellower than cheeses from silage- and hay-fed cows, which coincided with the color intensity perceived by the panelists. Moreover, cheeses from cows fed fresh herbage had more intense barnyard and dry fruit flavors, were perceived as creamier and having less lactic odor, and exhibited more fat exudation than those from cows fed conserved herbage. Only a few differences were observed in milk and cheeses from hay-fed compared with silage-fed cows, and those differences were far less pronounced than those of milk and cheeses from cows fed fresh herbage. In conclusion, the present study did not substantiate assumptions of clear sensory differences of milk and uncooked pressed cheese from hay-fed compared with silage-fed cows. For the first time, this study reports that the global flavor intensity of cheeses from indoor green-fed cows is similar to that of cheeses derived from cows fed conserved forages, whereas cheeses from grazing cows have the greatest global flavor intensity.

Claimed effects, outcome variables and methods of measurement for health claims proposed under European Community Regulation 1924/2006 in the framework of protection against oxidative damage and cardiovascular health.

BACKGROUND AND AIMS: The high number of negative opinions from the European Food Safety Authority (EFSA) to the requests for authorization of health claims is largely due to the design of human intervention studies, including the inappropriate choice of outcome variables (OVs) and of their methods of measurement (MMs). The present manuscript reports the results of an investigation aimed to collect, collate and critically analyse the information in relation to claimed effects, OVs and MMs, in the context of protection against oxidative damage and cardiovascular health compliant with Regulation 1924/2006. METHODS AND RESULTS: Claimed effects, OVs and the related MMs were collected from EFSA Guidance documents and applications for authorization of health claims under Articles 13.5 and 14. The OVs and their MMs were evaluated only if the claimed effect was sufficiently defined and was considered beneficial by EFSA. The collection, collation and critical analysis of the relevant scientific literature consisted in the definition of the keywords, the PubMed search strategies and the creation of databases of references. The critical analysis of the OVs and their MMs was performed on the basis of the literature review and was aimed at defining the appropriateness of OVs and MMs in the context of the specific claimed effects. CONCLUSIONS: The information provided in this document could serve to EFSA for the development of further guidance on the scientific requirements for health claims, as well as to the stakeholders for the proper design of human intervention studies aimed to substantiate such health claims.

Development and validation of an high performance liquid chromatography-tandem mass spectrometry method for the determination of imatinib in rat tissues.

An high performance liquid chromatography-tandem mass-spectrometry (HPLC-MS/MS) method was developed and validated for the determination in rat heart and liver of the tyrosine kinase inhibitor imatinib (IM), an anticancer drug approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Extraction of the drug from tissues was performed by solvent extraction and the obtained extracts were analyzed by HPLC-MS/MS in selected reaction monitoring mode. The developed method was validated according to the criteria for bioanalytical method, showing good performances in terms of lower limit of quantification (LLOQ=0.02µgml(-1)), linearity (R(2)=0.998), repeatability (RSD<3%), reproducibility (RSD<13%) and recovery (RR>89%). The developed method was then applied to the analysis of heart and liver of rats treated with different doses of IM, with and without the simultaneous administration of carvedilol, a beta-blocking agent with cardioprotective effect, in order to evaluate tissue levels of the tyrosine kinase inhibitor. The obtained results revealed that the amount of IM in the rat heart was significantly affected by the administered dose, whereas carvedilol had no effect on IM concentrations. Thus, we have developed a method that allows the detection of IM traces in complex tissues such as the heart and liver and that may be proposed for the determination of the drug in other clinically relevant biological samples.

Fully automated solid-phase microextraction-fast gas chromatography-mass spectrometry method using a new ionic liquid column for high-throughput analysis of sarcosine and N-ethylglycine in human urine and urinary sediments.

A fully automated, non invasive, rapid and high-throughput method for the direct determination of sarcosine and N-ethylglycine in urine and urinary sediments using hexyl chloroformate derivatization followed by direct immersion solid-phase micro extraction and fast gas chromatography-mass spectrometric analysis was developed and validated. The use of a new ionic liquid narrow bore column, as well as the automation and miniaturization of the preparation procedure by a customized configuration of the utilized XYZ robotic system, allowed a friendly use of the GC apparatus achieving a quantitation limit of 0.06 µg L(-1) for sarcosine, good repeatability with CV always lower than 7% and reduced analysis times useful for point-of-care testing. The method was then applied for the analysis of 56 samples of urine and urinary sediments in healthy subjects, in those with benign prostatic hypertrophy and in patients with clinically localized prostate cancer. The results obtained showed that the medians of sarcosine/creatinine in urine were 103, 137 and 267 µg g(-1) respectively, thus assessing the potential use of sarcosine as urinary biomarker for prostate cancer detection. The highest values of sensitivity (79%) and specificity (87%) were obtained in correspondence of a cut-off value of 179 µg sarcosine(g creatinine)(-1), thus by using this cut-off threshold, sarcosine was significantly associated with the presence of cancer (p<0.0001). Finally, ROC analyses proved that the discrimination between clinically localized prostate cancer and patients without evidence of tumor is significantly correlated with sarcosine.

Characterization of the volatile profile of thistle honey using headspace solid-phase microextraction and gas chromatography-mass spectrometry.

In this study, a headspace solid-phase microextraction method was developed for the characterization of the volatile fraction of thistle honey and compared with a dynamic headspace extraction method. A DVB/CAR/PDMS fibre was used. The effects of extraction time, equilibration time and salt addition on extraction yield were evaluated. The volatile fraction of seven Italian thistle honey samples was extracted under the optimized conditions and analyzed by gas chromatography-mass spectrometry. Characterization of the volatile profile was performed in terms of nature and relative amount of the extracted compounds. A total of 40 compounds, belonging to different chemical classes, were identified. The relative amounts of 16 compounds found in all the analyzed thistle honeys, i.e. nonanal, furfural, decanal, 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran, benzaldehyde, α-linalool, lilac aldehyde (isomer IV), hotrienol, phenylacetaldehyde, 4-oxoisophorone, benzyl alcohol, 2-phenylethanol, a not identified compound, octanoic acid, nonanoic acid and methyl anthranilate, were calculated and submitted to statistical analysis, in order to define for each compound a typical range. On the basis of the obtained data, a characteristic set of values was defined for thistle honey volatile fingerprint. The developed model proved to be effective in recognizing the botanical origin of thistle honey.

Fluorescence in situ hybridization to improve the diagnosis of endocarditis: a pilot study.

Infective endocarditis is a rare but life-threatening disease associated with high mortality. Early diagnosis of the causative microorganism is critical to patient outcome. However, conventional diagnostic methods are often unsatisfactory in achieving this goal. As a proof of concept, we applied fluorescence in situ hybridization (FISH) for detection and identification of bacteria in histological sections of heart valves. Biopsy specimens from 54 suspected endocarditis patients were obtained during valve surgery and analysed via FISH. Specimens were screened with a probe panel that identifies the most common bacteria implicated in endocarditis. Results were compared with those of culture-based diagnostics and clinical data. Discrepant results were subjected to comparative sequence analysis of PCR-amplified 16S rRNA genes. FISH detected bacteria in 26 of the 54 heart valves. FISH allowed successful diagnosis of infective endocarditis in five of 13 blood culture-negative cases and in 11 of 37 valve culture-negative cases, showing the bacteria within their histological context. This technique allows the simultaneous detection and identification of microorganisms at the species or genus level directly from heart valves and might be a valuable tool for diagnosis of endocarditis.

A novel headspace solid-phase microextraction method using in situ derivatization and a diethoxydiphenylsilane fibre for the gas chromatography-mass spectrometry determination of urinary hydroxy polycyclic aromatic hydrocarbons.

An innovative and simple headspace solid-phase microextraction method using a novel diethoxydiphenylsilane fibre based on in situ derivatization with acetic anhydride was optimized and validated for the gas chromatography-mass spectrometry determination of some monohydroxy metabolites of polycyclic aromatic hydrocarbons, namely 1-hydroxynaphthalene, 2-hydroxynaphtalene, 9-hydroxyfluorene, 2-hydroxyfluorene and 1-hydroxypyrene at trace levels in human urine. Enzymatic hydrolysis was applied before derivatization, whereas extraction conditions, i.e. the effects of time and temperature of extraction and salt addition were investigated by experimental design. Regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction response. These conditions were found in correspondence of an extraction temperature of 90 degrees C, an extraction time of 90 min and 25% NaCl added to urine samples. The capabilities of the developed method were proved obtaining limit of quantitations in the 0.1-2 microg/l range, thus allowing the bio-monitoring of these compounds in human urine. A good precision was observed both in terms of intra-batch and inter-batch repeatability with RSD always lower than 14%. Recoveries ranging from 98(+/-3) to 121(+/-1)% and extraction yields higher than 72% were also obtained. Finally, the analysis of urine specimens of coke-oven workers revealed analytes' concentrations in the 2.2-164 microg/l range, proving the exposure to PAHs of the involved workers.

Differentiation of the volatile profile of microbiologically contaminated canned tomatoes by dynamic headspace extraction followed by gas chromatography-mass spectrometry analysis.

The aromatic profile of microbiologically contaminated canned tomatoes was analyzed by the dynamic headspace extraction technique coupled with gas chromatography-mass spectrometry. Canned tomatoes contaminated with Escherichia coli, Saccharomyces cerevisiae and Aspergillus carbonarius were analyzed after 2 and 7 days. About 100 volatiles were detected, among which alcohols, aldehydes and ketones were the most abundant compounds. Gas chromatographic peak areas were used for statistical purposes. First, principal component analysis was carried out in order to visualize data trends and clusters. Then, linear discriminant analysis was performed in order to detect the set of volatile compounds ables to differentiate groups of analyzed samples. Five volatile compounds, i.e. ethanol, beta-myrcene, o-methyl styrene, 6-methyl-5-hepten-2-ol and 1-octanol, were found to be able to better discriminate between uncontaminated and contaminated samples. Prediction ability of the calculated model was estimated to be 100% by the "leave-one-out" cross-validation. An electronic nose device was then used to analyze the same contaminated and not contaminated canned tomato samples. Preliminary results were compared with those obtained by dynamic headspace gas chromatography-mass spectrometry, showing a good agreement.

Hydroxyestrogens inhibit angiogenesis in swine ovarian follicles.

The rapid, controlled, and cyclical nature of angiogenesis in the ovarian follicle suggests the potential for sex steroids to influence neovascularization. Angiogenesis is regulated by a local balance between the levels of endogenous stimulators and inhibitors. Multiple lines of evidence suggest that estrogens stimulate angiogenesis via effects on endothelial cells. However, it is of outstanding value to investigate the negative control of this process. Since the main estrogen metabolites, 2-hydroxyestradiol and 4-hydroxyestradiol (4-OHE2) have been demonstrated to function as anti-estrogen in several estrogen-dependent organs; the aim of this study was to investigate their potential involvement in the modulation of follicular angiogenesis. Hydroxyestrogens were quantified in swine follicular fluid and their effects were studied on granulosa cell vascular endothelial growth factor (VEGFA) production and tested in an angiogenesis bioassay. Our study documents that these molecules are physiologically present in swine follicular fluid and their concentrations significantly (P<0.001) increase during follicle development. Moreover, angiogenesis bioassay revealed that both hydroxyestrogens significantly (P<0.001) inhibited new vessel growth. We evidenced that the most potent negative effect is mediated by 4-OHE2. The anti-angiogenic potential of this molecule is also supported by its ability to inhibit (P<0.001) VEGFA production by granulosa cells. Increased knowledge in this area is of utmost importance for future therapeutic options to contrast infertility disorders associated with aberrant angiogenesis; this would be also very useful for the treatment of diseases characterized by disregulated angiogenesis and vascular regression.

Development and validation of an inductively coupled plasma mass spectrometry method with optimized microwave-assisted sample digestion for the determination of platinum at ultratrace levels in plasma and ultrafiltrate plasma.

A highly sensitive inductively coupled plasma mass spectrometry (ICP-MS) method with microwave-assisted sample digestion for the determination of total platinum in rat whole and ultrafiltrate plasma was developed and validated. A first step of this study concerned the optimization of the mineralization procedure, in order to obtain good extraction recovery (higher than 90%) and repeatability (less than 6%) and the absence of matrix effect. ICP-MS analysis was then performed using the "hot plasma/protective ion extraction" mode, achieving high sensitivity and very high signal/noise ratio. Iridium was added as internal standard. The method was then submitted to validation, performed according to the FDA Bioanalytical Validation Methods guidelines and to the Eurachem guide. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy and stability. An instrumental LOQ of 1.9ngL(-1), corresponding to a concentration of 955ngL(-1) in matrix under the adopted conditions, was obtained, allowing the quantitative analysis of Pt ultratraces. Instrumental linearity was verified in the range 1.9-14,000ngL(-1), corresponding to a concentration range from 955ngL(-1) to 6825microgL(-1) in matrix. Accuracy was evaluated by analyzing control samples for both matrices at different concentration levels; a good agreement (<15%) was obtained. Sample stability was tested by analyzing control samples maintained for 4h at room temperature or submitted to three freezing-thawing cycles. Finally, the developed method was applied to the analysis of plasma and ultrafiltrate plasma of rats treated with oxaliplatin-base drug, thus demonstrating its reliability in pharmacokinetic studies.

Experimental design for the optimization of the extraction conditions of polycyclic aromatic hydrocarbons in milk with a novel diethoxydiphenylsilane solid-phase microextraction fiber.

An innovative solid-phase microextraction coating based on the use of diethoxydiphenylsilane synthesized by sol-gel technology was used for the determination of polycyclic aromatic hydrocarbons at trace levels in milk. The effects of time and temperature of extraction and acetone addition were investigated by experimental design. Regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction response. The capabilities of the developed fiber were proved obtaining limit of quantitation values in the low microg/l range, enabling the direct analysis of complex matrices like milk and a complete desorption of high-boiling compounds without carryover effects.

Porcine follicular fluids: comparison of solid-phase extraction and matrix solid-phase dispersion for the GC-MS determination of hormones during follicular growth.

The capabilities of solid-phase extraction (SPE) and matrix solid-phase dispersion (MSPD) for the determination of the hormones 17beta-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol and 2-methoxyestradiol by gas chromatography-mass spectrometry (GC-MS) in a very complex matrix like porcine follicular fluids were compared, thus proving the highest effectiveness of the SPE technique. Validation was carried out in terms of limit of quantitation (LOQ), precision, accuracy, recovery and stability. LOQ values in the low microg kg(-1) were achieved, with all the other parameters satisfying the acceptance criteria for the validation of bioanalytical methods. The applicability of the method to the determination of the hormones in porcine follicular fluids was demonstrated, thus allowing to observe an increase of the concentration of the hormones during the follicular growth.

Characterization of the aromatic profile for the authentication and differentiation of typical Italian dry-sausages.

In order to chacterize two kinds of typical Italian dry-sausages, namely "Salame Mantovano" and "Salame Cremonese", the volatile composition was determined for seven samples of "Salame Mantovano" and for five samples of "Salame Cremonese". The study was performed by the dynamic headspace extraction technique (DHS) coupled with gas chromatography-mass spectrometry (GC-MS). Among the 104 volatiles identified, terpenes, aldehydes, ketones and alcohols represented the most abundant compounds. Peak area data for all the substances from the above mentioned group was used for statistical purposes. Firstly, principal component analysis (PCA) was carried out in order to visualize data trends and to detect possible clusters within samples. Then, linear discriminant analysis (LDA) was performed in order to detect the volatile compounds able to differentiate the two kinds of sausages investigated. The data obtained by GC-MS shows that the most important contributions to the differentiation of the two kinds of typical Italian salami were seven volatile compounds, i.e. 3-methylbutanal, 6-camphenol, dimethyl disulfide, 1-propene-3,3'-thiobis, ethyl propanoate, 1,4-p-menthadiene and 2,6-dimethyl-1,3,5,7-octatetraene. Prediction ability of the calculated model was estimated to be 100% by the "leave-one-out" cross-validation.

Orthotopic heart transplantation with concurrent coronary artery bypass grafting or previous stent implantation.

We describe three cases of donor hearts with preexisting coronary artery disease already diagnosed prior to transplantation: two were treated by coronary artery bypass grafting during the transplant procedure and one by angioplasty with stenting during the donor screening angiography. All three donor organs would otherwise have been rejected, depriving potential recipients of organ transplantation. All patients had an uneventful post-operative course with follow-up completed 22, 40 and 43 months after orthotopic transplantation showing patency of the stent and bypass grafts in the early (1 and 9 months) and late (22, 24 and 37 months) coronary angiography. Our results suggest that in this era of acute organ shortage donor hearts requiring bypass or stenting, which form a small but significant donor subgroup, can be used effectively and safely when matched to the recipients' age and medical condition.

Use of experimental design for the purge-and-trap-gas chromatography-mass spectrometry determination of methyl tert.-butyl ether, tert.-butyl alcohol and BTEX in groundwater at trace level.

An efficient method for the simultaneous determination of methyl tert.-butyl ether, tert.-butyl alcohol, benzene, toluene, ethylbenzene and xylene isomers in groundwater by purge-and-trap-gas chromatography-mass spectrometry was developed and validated. Experimental design was used to investigate the effects of temperature of extraction, time of extraction and percentage of salt added to the water samples. Regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction yield. Validation was carried out in terms of limits of detection (LOD), limits of quantitation (LOQ), linearity and precision. LOD values ranging from 2.6 to 23 ng l(-1) were achieved, whereas linearity was statistically verified over two orders of magnitude for each compound. Precision was evaluated testing two concentration levels. Good results were obtained both in terms of intra-day repeatability and intermediate precision: RSD% lower than 4.5% at the highest concentration and lower than 13% at the lowest one were calculated for intra-day repeatability. A groundwater sample suspected of contamination by leaking underground petroleum storage tanks was analysed and some of the analytes were detected and quantitated.

Surgical management of acute aortic root endocarditis with viable homograft: 13-year experience.

OBJECTIVE: Cryopreserved homograft valves have been used for acute infective aortic root endocarditis with great success but it is compounded by its availability in all sizes. The long-term clinical results of geometric mismatched homografts are not well defined and addressed. METHODS: Over a 15-year period (April 1986-June 2001), 816 patients presented with active infective endocarditis. One hundred and eighty-two of the patients aged between 9 and 78 years (mean: 51.0 +/- 1.13 years) consisting of 142 males and 40 females received homograft aortic valves. One hundred and ten patients were in NYHA functional class III and 72 in class IV and in cardiogenic shock. Of the patients, 2.7% suffered from septic embolism. One hundred and twenty-four (68.1%) patients presented with periannular abscesses and 58 (31.9%) with no abscess while 107 native valve (NVE) and 75 prosthetic valve (PVE) endocarditis were diagnosed preoperatively by transesophageal echocardiography (TEE) and confirmed intraoperatively. Freehand subcoronary implantation (FSCI) was used in 106 patients and root replacement in 76 patients. RESULTS: The operative death was 8.5% and for patients in NYHA functional class IV and in cardiogenic shock was 14.5%. Late mortality rate was 7.9%. Patient survival after discharge from hospital at 1 year was 97% and at 10 years was 91%, respectively. Thirty-one (22.1%) patients underwent reoperation after 1.7 years (mean) with two deaths (6.4%). Early (< or = 60 days) and late reinfection rate was 2.7 and 3.6%, respectively. Freedom from reoperation for matched and undersized homografts at 10-13 years was 85 and 55%, respectively. The univariate model identified undersized homograft (P=0.002), FSCI (P=0.09) and reinfection (P=0.0001) as independent risk factors for developing early and late valve dysfunction resulting in reoperation and homograft explant. CONCLUSION: Early aggressive valve replacement with homograft for active infective aortic root endocarditis with periannular abscesses is more successful than delayed last resort surgery. Homografts exhibit excellent clinical performance and durability with a low rate of reinfection, if properly inserted. Undersized homograft is an incremental risk factor for early and late reoperation.

A comparison of prototype compound parabolic collector-reactors (CPC) on the road to SOLARDETOX technology.

Solar photocatalytic detoxification of non-biodegradable chlorinated hydrocarbon solvents (NBCS) is carried out in different concentrating and non concentrating devices using TiO2 as a photocatalyst fixed on the inner surface of the reaction tubes or as a slurry catalyst which has to be removed from the treated water. The reaction is most effective using 200 mg/l of TiO2 as a slurry in a non concentrating CPC reactor. The concentrating parabolic trough reactor has a poor activity because of its minor irradiated reactor surface. Catalyst coated glass tubes are less efficient then the used slurry catalyst. Their advantage is that no catalyst has not to be removed from the treated water and there is no loss of activity during treatment. Yet their physical stability is not sufficient to be competitive to the slurry catalyst. Nevertheless the degradation results are very promising and will possibly lead to commercial applications of this technology.