Paola Tolu

Assessment of reference ranges for blood Cu, Mn, Se and Zn in a selected Italian population

The reference ranges for blood Cu, Mn, Se and Zn of 215 adult subjects non-occupationally exposed and living in the Nuoro province (Sardinia, insular Italy) were assessed. Metals were determined by sector field inductively coupled plasma mass spectrometry after microwave-assisted acid digestion of blood. The blood reference ranges estimated as P5-P95 percentiles (geometric mean, GM) were 776-1495μg/L (1036μg/L) for Cu; 4.73-17.0μg/L (8.91μg/L) for Mn; 106-185μg/L (140μg/L) for Se and 4686-8585μg/L (6418μg/L) for Zn. These results were then stratified for sex, age, alcohol consumption, smoking habit and living area. The GM value of Cu was significantly higher in females (1127μg/L) than in males (957μg/L). Age, alcohol intake, smoking habit and residential area did not influence blood Cu. The GM of Mn was significantly higher in females (9.98μg/L) respect to males (8.01μg/L) and in drinkers (9.67μg/L) compared to non-drinkers (8.38μg/L). The other variables did not change the Mn data. Selenium concentrations did not differ significantly as a function of individuals factors. With reference to Zn, males had GM of 6804μg/L and females of 6031μg/L, and more blood Zn was found in subjects consuming alcohol (6618μg/L) respect to abstainers (6155μg/L). In addition, blood Zn was not affected by age, smoking habit and place of living.

Reference intervals for blood Cd and Pb in the general population of Sardinia (Italy)

The reference values (RVs) for blood Cd and Pb of 215 adult subjects non-occupationally exposed and living in Sardinia (insular Italy) were assessed. Age, sex, smoking, alcohol drinking and living area were used to stratify the reference group. After collection from volunteers, samples were acid digested in a microwave oven and metals were determined by sector field inductively coupled plasma mass spectrometry. The RVs expressed as 5th-95th percentiles (geometric mean, GM) were 0.24-1.82 μg/l (0.53 μg/l) for blood Cd and 13.2-87.3 μg/l (33.4 μg/l) for blood Pb. Females had GM levels of Cd (0.58 μg/l) higher than males (0.49 μg/l); subjects aged <40 years had less Cd (0.44 μg/l) than old subjects (>60 years; 0.56 μg/l); Cd in smokers (1.23 μg/l) was 3-times higher than in non-smokers (0.42 μg/l) and correlated with the number of cigarettes per day. The alcohol intake and place of living did not influence blood Cd. The GM values of blood Pb in males (44.4 μg/l) were higher than in females (24.7 μg/l); subjects less than 40 years-old (27.5 μg/l) showed lower Pb than elderly individuals (>60 years, 41.2 μg/l); drinkers (42.2 μg/l) had Pb 2-times higher than non-drinkers (24.4 μg/l). Blood Pb was not significantly affected by smoking and place of living. As revealed by multiple linear regression, the predictor variables were, in order of weight, smoke ≫ age for blood Cd levels, and sex = age ≫ alcohol for blood Pb levels.

Diet and nutrients are contributing factors that influence blood cadmium levels.

Studies suggested the intake of Cd from diet can be approximately equivalent to that from smoking. Moreover, a mutual metabolic influence between Cd and nutrients has been reported. The purpose of this study was to evaluate the relationship between blood cadmium concentration (BCdC) and food consumption, nutrients intake (Ca, Fe, Zn, vitamin C, and vitamin D), tobacco smoking, and some other variables (age, body mass index, and residence) in 243 adults living in the Italian island of Sardinia (Sassari Province). Specifically, we hypothesized that offal consumption contributes to Cd intakes and blood levels. The BCdC was quantified by graphite furnace atomic absorption spectrometry, and information on personal data was collected through questionnaires. Smoke significantly contributed to the BCdC (P < .001). Nonsmoker subjects who eat offal showed significantly higher BCdC (P = .04). Moreover, slightly higher BCdCs were also observed in nonsmoker subjects who eat rice, fish, and bread. The BCdC positively correlated with age of subjects (r = 0.144; P = .025) and offal daily intake in nonsmokers (r = 0.393; P < .001). The intake of Ca was negatively correlated (r = -0.281; P = .001) with the BCdC in females. The multiple linear regression analysis showed smoking > consumption of offal > body mass index ≈ age as the most important risk factors for the BCdC in the selected population.

Heavy Metals and Multiple Sclerosis in Sardinian Population (Italy)

The understanding of factors involved in the etiopathogenesis of Multiple Sclerosis (MS) is very important, especially in Sardinia (insular Italy) where one of the highest MS incidences in the world is consistently reported over time. There is evidence that some metals are implicated in the MS course and disability, and, a study was carried out to assess the concentration of Al, Cd, Cu, Fe, Mn, Pb, and Zn in the cerebrospinal fluid (CSF) of 51 Sardinian subjects (29 consecutive patients and 22 controls). In fact, any unbalance in the homeostasis of metals in the CSF might predispose to the cellular brain metal transport and accumulation giving rise to pathogenic lesions terminating in neurodegeneration. Samples were diluted with water and metals were quantified by sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). Results revealed that none of the metals quantified in the CSF were able to distinguish the MS cases from controls and the different MS forms. Furthermore, sex, age, coffee, alcohol intake, and smoking did not represent risk factors for the disease. In light of this outcome, the reasons of the high incidence of MS in Sardinia may be more likely addressed to the susceptible genetic background that is easily triggered by other environmental agents. The results are the first data set on the elemental profile in the CSF of Sardinian patients and can be used for comparisons with possible abnormal contents in other pathologies.

Biological markers in CSF of Sardinian patients with multiple sclerosis

Multiple Sclerosis (MS) is an inflammatory disease of the Central Nervous System (CNS). The lesions appear in seemingly random areas of the CNS white matter. Theories try to combine the known data into plausible explanations, but none has proved definitive so the specific cause of MS is not fully understood. This inflammatory response may be triggered by genetic, environmental, and viral factors that initiate demyelination. The different courses of the disease, both within an individual and within the whole population, principally differ in their timing, location and severity. There is a very specific geographic distribution of this disease around the world. A significantly higher incidence of the disease is found in Sardinia. Sardinia represents an ideal large data set for MS aetiopathogenic studies. Biomarkers in cerebrospinal fluid (CSF) could help to predict and monitor neurological decline in people with MS. Neurons and glial cells exhibit a remarkable diversity of shapes. The evidence indicates that the shape of cells in the nervous system is closely connected to their functions. The aim of this study is to determine if CNS-derived proteins present in the CSF of MS patients reflect different pathologic processes of MS and if these proteins could be useful as biologic markers of disease activity. In particular we have studied the following proteins: NFL, GFAP and the isoforms II and III of tubulin β. CNS cytoskeleton proteins concentration was performed by dot blot technique. The results of the concentration of the four proteins are expressed in Arbitrary Unit (AU) that is the relationship between the optical density of the spots obtained and the concentration of the protein. The concentration values of the 4 proteins of cytoskeleton are higher in all MS patients compared to both control groups. In particular the NF increase significantly in the RRMS group than in the RPMS group, while the values of GFAP are slightly increase in RRMS group. About tubulins, while the values of tubulin β isophorm II are almost identical in 2 groups of MS patients, the tubulin β isophorm III shows levels higher in the group RPMS than in the group RRMS.

Electronic microscopy in MCF7 cells : Cd - 5-Fu effects

Cadmium (Cd) is known as a highly toxic metal wich represents a major hazard to the environment The extremely long biological half life (30-35 years) makes it a cumulative toxin; therefore long-term past exposure could still result in direct toxic effects. The toxicity of this metal contributes to a large variety of health conditions, including major diseases such as heart disease, cancer and diabetes. The fluoropyrimidines, especially 5-Fluorouracil (5-Fu) are antimetabolite inhibitors of the novo purine and pyrimidines syntheses. 5-Fu play an important role in standard chemotherapy protocols for a range of solid tumors, including breast and colorectal cancers. Breast cancer represent the most common tumor of female in many industrialized countries. The MCF7 human breast cancer cell line had been used as an excellent experimental model to improve the efficacy of different therapies before use in patients. In order to elucidate the mechanism of both Cadmium and 5-Fu effects, we compared the morphological transformations with observation by TEM and SEM electronic microscopy. MCF7 cells were grown at 37° C in an atmosphere containing 5% of Co2 with RPMI 1640 Medium supplemented with 10% of fetal bovine serum. For Elettronic Microscopy, MCF7 cells were treated with Cd (5uM, 20uM, 40uM) and 5-Fu (1,5uM, 50uM) for different time of incubation (6h, 24h, 48h). The cells, to be observed by TEM, were fixed in 2% glutharaldehyde and postfixed in 1% OsO4, washed and then embedded in 1.5% low-melting-point agarose. Samples were stained, after ultrathin sections, with 4% uranyl acetate and lead citrate. Culture cells to be observed by SEM were fixed in 2.5% glutaraldehyde, then washed and post fixed in 1% OsO4. Samples were dehydrated in ethanol, incubated in hexamethyldisilazane and examined in low vacuum using a SEM FEI Quanta 200. In the cells treated with Cd already to small concentrations and brief times of incubation it increases the number of the vesicles, probable peroxisoms that begins to polish up the cells from the cadmium. To tall concentrations of Cd (20uM -40uM) we note notable increase of the cytological disorder, the nucleuses are very evident and globular. The addition of 5-fu (1,5uM) to cells treated with Cd (5 uM20 uM) brings to a reduction of the number of the secretion’s vesicles and the number of filopods, in fact to 48h the cells are introduced flat with a smoother cellular surface. Under extreme conditions (cd 40, 48h), the addition of the anti-tumoral substance doesn’t meaningfully modify the cellular disorder observed with only Cd.