Assessing two-way interactions between cells and inorganic nanoparticles.

A safe and effective use of nanoparticles in biology and medicine requires a thorough understanding, down to the molecular level, of how nanoparticles interact with cells in the physiological environment. This study evaluated the two-way interaction between inorganic nanomaterials (INMs) and cells from A549 human lung carcinoma cell line. The interaction between silica and zinc oxide INMs and cells was investigated using both standard methods and advanced characterization techniques. The effect of INMs on cell properties was evaluated in terms of cell viability, chemical modifications, and volume changes. The effect of cells and culture medium on INMs was evaluated using dynamic light scattering (DLS), scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS), high performance liquid chromatography (HPLC), gas chromatography-mass spectroscopy (GC-MS), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). No cytotoxic effect was detected in the case of silicon oxide INMs, while for high doses of zinc oxide INMs a reduction of cell survival was observed. Also, increased cell volume was recorded after 24 h incubation of cells with zinc oxide INMs. A better dimensional homogeneity and colloidal stability was observed by DLS for silicon oxide INMs than for zinc oxide INMs. SEM-EDS analysis showed the effectiveness of the adopted dispersion procedure and confirmed in the case of zinc oxide INMs the presence of residual substances derived from organosilane coating. HPLC and GC-MS performed on INMs aqueous dispersions after 24 h incubation showed an additional peak related to the presence of an organic contaminant only in the case of zinc oxide INMs. FTIR Chemical Imaging carried out directly on the cells showed, in case of incubation with zinc oxide INMs, a modification of the spectra in correspondence of phospholipids, nucleic acids and proteins characteristic absorption bands when compared with untreated cells. Overall, our results confirm the importance of developing new experimental methods and techniques for improving the knowledge about the biosafety of nanomaterials.

Determination of carbonyl compounds in exhaled breath by on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry.

A reliable method for the determination of carbonyl compounds in exhaled breath based on on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry is described. The analytical performances were optimized for a mixture of C2-C9 aldehydes and C3-C9 ketones, particularly interesting for clinical applications, by using an internal standard and applying a 23 full factorial design. A volume of sample (250 ml) was loaded at 50 ml min-1 into a Tenax GR sorbent tube containing 130 nmol of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride. All compounds showed a limit of detection lower than 200 pptv. The yield of the derivatization procedure was normalized by adding to the sample a known amount of 6D-acetone as an internal standard. This allowed halving the relative standard deviation to 10% and 15% for the mono-and di-carbonyl compounds, respectively, thus improving reliability. The optimized method was applied to the determination of carbonyl compounds in 12 breath samples collected from four patients suffering from heart failure during hospitalization.

Determination of volatile organic compounds in exhaled breath of heart failure patients by needle trap micro-extraction coupled with gas chromatography-tandem mass spectrometry.

The analytical performances of needle trap micro-extraction (NTME) coupled with gas chromatography-tandem mass spectrometry were evaluated by analyzing a mixture of twenty-two representative breath volatile organic compounds (VOCs) belonging to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics and sulfurs). NTME is an emerging technique that guarantees detection limits in the pptv range by pre-concentrating low volumes of sample, and it is particularly suitable for breath analysis. For most VOCs, detection limits between 20 and 500 pptv were obtained by pre-concentrating 25 ml of a humidified standard gas mixture at a flow rate of 15 ml min-1. For all compounds, inter- and intra-day precisions were always below 15%, confirming the reliability of the method. The procedure was successfully applied to the analysis of exhaled breath samples collected from forty heart failure (HF) patients during their stay in the University Hospital of Pisa. The majority of patients (about 80%) showed a significant decrease of breath acetone levels (a factor of 3 or higher) at discharge compared to admission (acute phase) in correspondence to the improved clinical conditions during hospitalization, thus making this compound eligible as a biomarker of HF exacerbation.

Comparison of sampling bags for the analysis of volatile organic compounds in breath.

Nalophan, Tedlar and Cali-5-Bond polymeric bags were compared to determine the most suitable type for breath sampling and storage when volatile organic compounds are to be determined. Analyses were performed by thermal desorption gas chromatography mass spectrometry. For each bag, the release of contaminants and the chemical stability of a gaseous standard mixture containing eighteen organic compounds, as well as the CO2 partial pressure were assessed. The selected compounds were representative of breath constituents and belonged to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics, sulfurs and esters). In the case of Nalophan, the influence of the surface-to-volume ratio, related to the bag's filling degree, on the chemical stability was also evaluated. Nalophan bags were found to be the most suitable in terms of contaminants released during storage (only 2-methyl-1,3-dioxalane), good sample stability (up to 24 h for both dry and humid samples), and very limited costs (about 1 € for a 20 liter bag). The (film) surface-to-(sample) volume ratio was found to be an important factor affecting the stability of selected compounds, and therefore we recommended to fill the bag completely.

Post-operative elimination of sevoflurane anesthetic and hexafluoroisopropanol metabolite in exhaled breath: pharmacokinetic models for assessing liver function.

Sevoflurane (SEV), a commonly used anesthetic agent for invasive surgery, is directly eliminated via exhaled breath and indirectly by metabolic conversion to inorganic fluoride and hexafluoroisopropanol (HFIP), which is also eliminated in the breath. We studied the post-operative elimination of SEV and HFIP of six patients that had undergone a variety of surgeries lasting between 2.5 to 8.5 h using exhaled breath analysis. A classical three compartments pharmacokinetic model developed for the study of environmental contaminants was fitted to the breath data. We found that SEV kinetic behavior following surgery (for up to six days) is consistent across all subjects whereas the production and elimination of HFIP varies to some extent. We developed subject specific parameters for HFIP metabolism and interpreted the differences in the context of timing and dose of anesthesia, type of surgery, and specific host factors. We propose methods for assessing individual patient liver function using SEV as a probe molecule for assessing efficiency of liver metabolism to HFIP. This work is valuable not only for the clinical study of metabolism recovery, but potentially also for the study of the interaction of other manufactured and environmental compounds with human systems biology in controlled exposure and observational studies.

Monitoring breath during oral glucose tolerance tests.

The evolution of breath composition during oral glucose tolerance tests (OGTTs) was analysed by thermal desorption/gas chromatography/mass spectrometry in 16 subjects and correlated to blood glucose levels. The glucose tolerance tests classified five of the subjects as diabetics, eight as affected by impaired glucose tolerance and three as normoglycaemic. Acetone levels were generally higher in diabetics (average concentration values: diabetics, 300 ± 40 ppbv; impaired glucose tolerance, 350 ± 30 ppbv; normoglycaemic, 230 ± 20 ppbv) but the large inter-individual variability did not allow us to identify the three groups by this parameter alone. The exhalation of 3-hydroxy-butan-2-one and butane-2,3-dione, likely due to the metabolization of glucose by bacteria in the mouth, was also observed. Future work will involve the extension of the analyses to other volatile compounds by attempting to improve the level of discrimination between the various classes of subjects.

Quantification of nitrite and nitrate in seawater by triethyloxonium tetrafluoroborate derivatization-headspace SPME GC-MS.

Triethyloxonium tetrafluoroborate derivatization combined with direct headspace (HS) or SPME-gas chromatography-mass spectrometry (GC-MS) is proposed here for the simultaneous determination of nitrite and nitrate in seawater at micromolar level after conversion to their corresponding volatile ethyl-esters (EtO-NO and EtO-NO(2)). Isotopically enriched nitrite [(15)N] and nitrate [(15)N] are employed as internal standards and for quantification purposes. HS-GC-MS provided instrumental detection limits of 0.07 µM NO(2)(-) and 2 µM NO(3)(-). Validation of the methodology was achieved by determination of nitrite and nitrate in MOOS-1 (Seawater Certified Reference Material for Nutrients, NRC Canada), yielding results in excellent agreement with certified values. All critical aspects connected with the potential inter-conversion between nitrite and nitrate (less than 10%) were evaluated and corrected for by the use of the isotopically enriched internal standard.

Evaluation of white matter damage in patients with Alzheimer's disease and in patients with mild cognitive impairment by using diffusion tensor imaging.

PURPOSE: The objective of this study was to evaluate white matter tissue damage in patients with Alzheimer's disease (AD) and in patients with mild cognitive impairment (MCI) using diffusion tensor imaging (DTI). MATERIALS AND METHODS: Forty-seven subjects were evaluated: 14 patients with AD, 15 with MCI and 18 healthy volunteers. All subjects were studied using conventional magnetic resonance imaging (MRI) and DTI (32 directions) with a 1.5 T magnet. Fractional anisotropy (FA) was measured in the following regions: frontal, occipital, parietal and temporal white matter and in the genu and splenium of the corpus callosum. The results were compared between the different groups and correlated with the Mini-Mental State Evaluation (MMSE) scores. RESULTS: A statistically significant difference was obtained between controls and MCI patients (p < 0.007) and between controls and AD patients (p < 0.05) with regard to FA of the white matter in the splenium. A statistically significant difference was obtained between controls and AD patients with regard to FA in the genu (p < 0.016). Moreover, there was a statistically significant difference between controls and AD patients considering the genu (p < 0.016) and the frontal white matter on the right side (p < 0.024). The MMSE scores correlated with the FA values measured in the genu, the splenium and frontal white matter on the right side. No significant differences were identified between patients with AD and those with MCI. CONCLUSIONS: DTI could be of value in the early detection of white-matter damage in patients with MCI and AD. The DTI values correlate with the neuropsychological tests.

Implementation of Fowler's method for end-tidal air sampling.

The design, realization and testing of a CO(2)-triggered breath sampler, capable of a separate collection of dead space and end-tidal air on multiple breaths, is presented. This sampling procedure has advantages in terms of the sample volume, insights regarding the origin of compounds, increased reproducibility and higher concentrations of compounds. The high quality of design and the speed of the components ensure a breath-by-breath estimate of dead volume, as well as the comfort and safety of the subject under test. The system represents a valid tool to contribute to the development of a standardized sampling protocol needed to compare results obtained by the various groups in this field.

Different perception of cognitive impairment, behavioral disturbances, and functional disabilities between persons with mild cognitive impairment and mild Alzheimer's disease and their caregivers.

Insight in dementia is a multifaceted concept and ability, which includes the persons' perception of their behavioral and cognitive symptoms and functional disability. This ability seems to deteriorate as dementia progresses. The aim of this study was to evaluate the level of insight in the cognitive, behavioral, and functional disorders in a group of persons with mild cognitive impairment (MCI) or mild AD (Alzheimer's disease) and to compare their perception of their illness with that of their caregivers. The study involved a group of 121 persons with MCI and mild AD and their caregivers. The persons with MCI and mild AD were administered the tests Mini-Mental State Examination, Instrumental Activities of Daily Living, Activities of Daily Living, Neuropsychiatric Inventory, Schedule for the Assessment of Insight, Clinical Insight Rating Scale, and a short interview. Major differences were identified between how the persons with MCI or mild AD and their caregivers perceived the persons' cognitive and behavioral disorders. The group with MCI or mild AD underestimated their deficits, which were considered serious and disabling by their caregivers.

The clinical interview and the doctor-patient relationship in telemedicine.

The doctor-patient relationship is one of the most complex interpersonal relationships, in that it involves individuals who are not on the same level, it has not been sought by both individuals, it is emotionally loaded and it requires close mutual co-operation towards a shared goal. A well-managed clinical interview lays the grounds for a good doctor-patient relationship which in turn leads to further advantages. In a classical ambulatory setting the doctor-patient relationship should take into account a number of context variables: physical environment, doctor's clothing and instrumental vs. affective behavior. The classical medical consultation takes place with the doctor and patient facing each other in the same room. Human contact, interaction and communication condition the course and treatment of the illness. This classical setting has been radically changed with the advent of videoconferencing. Video-communication reduces personal contact with the general doctor or specialist. Medical philosophy and the social sciences have often discussed the risk of alienating or "objectifying" the patient. The setting of a videoconferencing interview is completely different as interaction is not direct but occurs via a technological system that enables an unusual form of visual and auditory interaction that may be more or less realistic depending on the technology employed. The growing use of telemedicine and videoconferencing has led to new reflections on the doctor-patient relationship in non-conventional settings and warrants in-depth studies on the dynamics of this form of consultation so as to increase the patients' level of satisfaction in telemedicine. The next step will be to verify experimentally and with adequate tools what the demands of patients are in a videoconferencing setting.

An open-label, comparative study of rivastigmine, donepezil and galantamine in a real-world setting.

OBJECTIVE: We analysed the effects of donepezil, rivastigmine and galantamine, prescribed for the treatment of Alzheimer disease in a real-world setting in Italy. METHODS: Outcome measures included the MiniMental State Examination (MMSE), the Alzheimer Disease Assessment Scale cognitive subscale (ADAS-cog), Instrumental Activity of Daily Living (IADL) and ADL scales. RESULTS: Seventy patients were treated with donepezil, 121 with rivastigmine and 51 with galantamine. At 6 months, rivastigmine-treated patients improved by 1.29 points from baseline on the ADAS-cog, while donepezil- and galantamine-treated patients showed 'no change' (changes of < 0.2 points). On the IADL, patients treated with rivastigmine, donepezil and galantamine showed decreases of 0.42, 0.58 and 0.75 points, respectively. On the ADL, donepezil- and galantamine-treated patients showed decreases of 0.44 and 0.86 points, respectively, while there was 'no change' with rivastigmine. On the MMSE, donepezil- and rivastigmine-treated patients showed 'no change' and galantamine-treated patients showed a mean decrease of 1.19 points. A subgroup analysis of 'pseudo-randomised' patients (rivastigmine, n = 63; donepezil, n = 55; galantamine, n = 51) supported the main findings. Side effects were similar (in type and frequency) in the three treatment groups. CONCLUSIONS: This is the first study to compare the effects of the three most commonly-used cholinesterase inhibitors on the MMSE, ADAS-cog, IADL and ADL. Limitations included its small population size, its open-label design, and the fact that patients were randomised only after the introduction of galantamine. There were no statistically significant differences between the three drugs at 3 months. While numerical trends were observed suggesting the effect of rivastigmine > donepezil > galantamine, larger, longer-term prospective studies are needed to confirm whether there are important differences in the long-term efficacy of the three drugs.

Stress in the caregivers of Alzheimer's patients: an experimental investigation in Italy.

In Italy, the family constitutes the major caregiving response to the needs of the elderly who are no longer self-sufficient. These caregivers are often wives or daughters who have chosen to keep the patient at home with them. On average, three-quarters of the caregiver's day is devoted to the patient, a proportion that tends to increase linearly as the disease progresses. The primary aim of our study was to describe a group of Italian caregivers of patients with a diagnosis of dementia to assess their levels of stress. We then correlated these caregivers with a number of sociodemographic variables and the patients' degree of cognitive impairment and independence in daily life activities. The study was conducted on a sample group of 236 caregivers of patients with a diagnosis of Alzheimer's disease (AD) according to the DSM-IV criteria. Each caregiver took part in a sociodemographic interview and filled in two questionnaires: the Caregiver Burden Inventory (CBI), to quantify the caregiving workload and the Brief Symptom Inventory (BSI), to assess the level of anxiety and depression. Patients were administered the Mini-Mental State Examination (MMSE) to evaluate their level of cognitive impairment and the Instrumental Activities of Daily Living (IADL) and Activities of Daily Living (ADL) tests to quantify their level of independence. The analysis of the results shows that the average Italian caregiver of an AD patient is a woman, approximately 60 years old. The majority of caregivers are spouses, followed by children. In general, these caregivers work at home, are housewives or retired, and are personally involved in caring for the patients. The greater the level of a patient's cognitive impairment and the less independent they are, the greater amount of care and supervision they require, leaving less free time for the caregiver; this leads to higher levels of anxiety related to caregiving. Toward the final stages of the disease, patient care tasks take up nearly all of the caregiver's free time, leaving them only about two hours per week for themselves or their friends. In conclusion, the management of dementia patients places a particular burden on the caregiver and involves several economic and social costs. The burden becomes heavier as the disease progresses, since the increase of cognitive disorders and the resulting reduction of independence in daily life, together with the onset of behavioral symptoms, heighten the caregiver's distress, anxiety, and depression. Establishing a network of community services able to alleviate the burden on families is therefore a priority. Parallels can be drawn between the stresses documented for American caregivers and their Italian counterparts.