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Ciriaco Carru

Publications and source records attributed to Ciriaco Carru.

33 records · Page 2Linked to original sources
PubMed2004

Plasma D-penicillamine redox state evaluation by capillary electrophoresis with laser-induced fluorescence.

D-Penicillamine (D-Pen) is a thiol drug used in the treatment of Wilson's disease, rheumatoid arthritis, metal intoxication and cystinuria. We have recently described a new capillary electrophoresis (CE) method to measure physiological thiols, in which separation of total plasma homocysteine, cysteine, cysteinylglycine, glutathione is achieved using the organic base N-methyl-D-glucamine in the run buffer. In this paper, we present an improvement of our method that allows a baseline separation of total plasma D-Pen from the physiological thiols. Moreover, reduced, free and protein-bound forms of drug are measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulphosalicylic acid (SSA). After derivatization with 5-iodoacetamidofluorescein (5-IAF), samples are separated and measured by capillary electrophoresis with laser-induced fluorescence in an uncoated fused-silica capillary (57 x 75 microm i.d.) using a phosphate/borate run buffer pH 11.4. In these conditions, the migration time of D-Pen is about 7 min and the time required for each analysis is roughly 10 min. The proposed method has been utilized to measure the various forms of the drug in a D-Pen administered Wilson's disease patient.

Fluorescence↗
PubMed2004

A new HPLC method for serum neopterin measurement and relationships with plasma thiols levels in healthy subjects.

Neopterin, a pyrazinopyrimidine compound, serves as a marker of cellular immune system activation, and it can be used as a prognostic predictor for certain types of diseases. We propose a new simple HPLC method to measure serum neopterin with highly sensitive fluorimetric detection. After TCA serum protein precipitation, the supernatant was diluted five times, injected into a C18 reversed-phase column and eluted at a flow rate of 1.5 mL/min by an isocratic water-acetonitrile (99:1) mobile phase. The natural fluorescence of the molecule was detected at excitation wavelength 353 nm and emission 438 nm. In these conditions the neopterin retention time was about 4 min. Our proposed method was compared with a validated chromatographic separation, and the obtained data of the serum neopterin from 35 healthy volunteers were analysed by Passing-Bablok regression and Bland-Altman test. Neopterin measurement in healthy subjects was also employed to investigate on its potential relationships with plasma thiols levels.

Adult↗
PubMed2004

Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis.

High concentrations of total plasma thiols such as cysteine and homocysteine are important risk factors for atherosclerosis and cardiovascular diseases. We have recently described a new laser-induced fluorescence capillary electrophoresis (CE-LIF) method to measure total plasma thiols, in which the baseline separation of cysteinylglycine, homocysteine, cysteine, and glutathione was achieved by adding the organic base N-methyl-D-glucamine to the run buffer. However, because the active fractions of homocysteine and cysteine responsible for vascular injuries are still unknown, research calls for a set up of methods able to analyze different forms of plasma thiols. In this paper, we present an improvement of our previous method that allows the measurement of different thiol forms. Total, reduced, and free thiols were measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulfosalicylic acid. After derivatization with 5-iodoacetamidofluorescein, samples were separated and measured by CE-LIF using a phosphate/borate buffer in the presence of 75 mmol/L N-methyl-D-glucamine. Oxidized thiols and protein bound thiols were calculated by difference, free minus reduced and total minus free form, respectively. Linearity, reproducibility, analytical recovery, and sensitivity were evaluated. The assay was used to measure the thiols redox status in 15 plasma samples from healthy volunteers.

Electrophoresis, Capillary↗
PubMed2004

Determination of creatinine in human serum by short-end injection capillary zone electrophoresis.

A new ultra-rapid free-solution capillary zone electrophoresis method to measure serum creatinine is presented. Procedural parameters such as injection mode, concentration and pH of phosphate running buffer and acidic deproteinization of serum samples were investigated. Short-end injection permits a decrease of the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, so reducing the migration distance. Thus, when a capillary with an effective length of 10.2 cm and a 40 mmol/L sodium phosphate buffer pH 2.35 was used, the obtained migration time of the creatinine peak was the shortest never described before, about 1.1 min. These conditions give a good reproducibility of the migration times (coefficient of variation, CV% < 0.5) and the peak areas (CV% < 2.8). Intra- and interassay CV were 3.06 and 6.26%, respectively, and analytical recovery was 99.4%. We compared our proposed method to Jaffé colorimetric assay, by measuring serum creatinine in 128 normal subjects. The obtained data were analyzed by the Passing and Bablok regression and Bland-Altman test. Creatinine concentration in healthy subjects was also used to investigate on its relationships with plasma thiols levels.

Creatinine↗
PubMed2004

Association between longevity and cytokine gene polymorphisms. A study in Sardinian centenarians.

BACKGROUND AND AIMS: Human longevity seems to be directly correlated with optimal functioning of the immune system, suggesting that some genetic determinants of longevity reside in those polymorphisms for the immune system genes which regulate immune-inflammatory responses, in particular cytokine gene polymorphisms. The frequency of -174C single nucleotide polymorphism (SNP) in the promoter region of the interleukin (IL)-6 gene is increased in Italian male centenarians. Moreover, the frequency of -1082G SNP at the 5' flanking region of the IL-10 gene coding sequence is increased among male centenarians, and that of +874A SNP at the interferon (IFN)-gamma gene was found more frequently in female centenarians. These findings indicate that different alleles at different cytokine gene codings for pro- (IL-6, IFN-gamma) or anti-inflammatory (IL-10) cytokines may affect the individual life-span expectancy, influencing the type and intensity of immune-inflammatory responses against environmental stressors. METHODS: In the present study, we analyzed these IL-6, IL-10 and IFN-gamma gene polymorphisms in 112 (36 male, 76 female) centenarians from the island of Sardinia, whose population shows a genetic background quite different from that of mainland Italy, as well as in 137 sixty-year-old controls from the same geographic area. RESULTS: No significant differences were observed on analyzing IL-6, IL-10 and IFN-gamma polymorphism frequencies among centenarians and controls, either on the whole and when the data were analyzed according to gender. CONCLUSIONS: These data indicate that gene polymorphisms of cytokines playing a major regulatory role in the inflammatory response do not affect life expectancy in the Sardinian population. Thus, cytokine/longevity associations have a population-specific component, being affected by the population-specific gene pool as well as by gene-environment interactions, behaving as survival rather than longevity genes.

Aged↗
PubMed2004

Identification of a geographic area characterized by extreme longevity in the Sardinia island: the AKEA study.

High prevalence and low female/male ratio for validated centenarians are observed in Sardinia and these findings appear to be thus far unique to this island. Moreover a specific region on the island is characterized by exceptional male longevity. We calculated the extreme longevity index (ELI), defined as the percentage of persons born in Sardinia between 1880 and 1900, who became centenarians. A gaussian smoothing method was used in order to identify the so-called 'Blue Zone', where longevity is concentrated in the central-eastern part of the island and covers all the mountainous areas of central Sardinia. The estimated life expectancy in the 'Blue Zone' is longer than in the remaining territory of the island especially for men and the male to female ratio among centenarians born in this area is 1.35 compared to 2.43 in the rest of Sardinia. The specific mechanism by which persons living in this territory were more likely to reach extreme longevity remains unknown but it is interesting to note that most of the 'longevity hot spots' identified in various regions of the world over the years have been located in mountainous geographical areas even if none of these longevity regions have been fully validated. An alternative and interesting hypothesis is that the high rate of inbreeding determined by frequent marriages between consanguineous individuals and low immigration rates have progressively decreased the variability of the genetic pool and facilitated the emergence of genetic characteristics that protect individuals from diseases that are major causes of mortality particularly in older individuals. Given the exceptionally high prevalence of male centenarians in the 'Blue Zone', it is reasonable to assume that either the environmental characteristics or the genetic factors, or both, exert their favorable effect more strongly in men than in women. Thus, the mechanism involved may be modulated by the hormonal milieu, or may be associated with genes located in the sex chromosomes.

Aged↗
PubMed2003

Optimization of the principal parameters for the ultrarapid electrophoretic separation of reduced and oxidized glutathione by capillary electrophoresis.

Several factors can influence the analytical efficiency and rapidity of the quantitative determination of erythrocyte glutathione by capillary zone electrophoresis (CZE). We optimized the time, efficiency and resolution of the electrophoretic separation of reduced (GSH) and oxidized (GSSG) glutathione by studying the influence of the most important factors affecting the separation, i.e. the pH and ionic strength of the electrolyte solution, the capillary length and temperature. Best results in the shortest time are obtained at 25 degrees C, using an uncoated 37 cm x 75 microm i.d. capillary and a 300 mmol/l borate buffer pH 7.8. These conditions give a good reproducibility of the corrected peak areas (R.S.D. 1.41 and 1.31%) and of the migration time (R.S.D. 0.22 and 0.26%) for GSH and GSSG, respectively. The high concentration buffer, besides permitting a good resolution of standard GSH and GSSG mix, allows also N-nitrosoglutathione detection. By shortening the capillary length to 27 cm, the separation time of GSH and GSSG can be further decreased to less than 60s. This shortened method, the most rapid described in literature, can detect and quantify GSH in red blood cells despite a loss of sensitivity. To compare the new method here described with the Beutler colorimetric method, the data relative to the GSH content of red blood cells from young normal subjects were analyzed by the Passing and Bablok regression and the Bland-Altman test.

Electrophoresis, Capillary↗
PubMed2003

PKC/Raf/MEK/ERK signaling pathway modulates native-LDL-induced E2F-1 gene expression and endothelial cell proliferation.

BACKGROUND AND OBJECTIVES: The interactions of low-density lipoprotein (LDL) with the endothelium are thought to play a major role in the development of atherosclerosis. Due to this reason, the molecular sequelae of events resulting from native LDL (N-LDL) interaction with human endothelial cells (HECs) are largely under investigation. METHODS AND RESULTS: Here, we report that the exposure of serum-free HECs to different concentrations of N-LDL-cholesterol (LDL-chol) elicited a time- and dose-dependent induction of DNA synthesis. The exposure of serum-free HECs to N-LDL was able to elicit a time- and dose-dependent increase of protein kinase C (PKC) activity that, along with the activation of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, leads to an increase in E2F-1 gene expression. In addition, the treatment of HECs with N-LDL was also able to induce both E2F-1 gene transcription and protein expression. These N-LDL-aroused responses were dramatically counteracted by PKC inhibition or down regulation. Similarly to what observed for Raf/MEK/ERK activation and E2F-1 gene expression, the inhibition of PKC as well as its down regulation, significantly lowered the DNA synthesis induced by N-LDL in serum-free HECs. CONCLUSIONS: These results suggest that the activation of PKC/Raf/MEK/ERK-mediated events controlling E2F-1 gene expression by N-LDL may represent an important mechanism in the regulation of HECs proliferation during normal and pathological processes.

Cell Cycle Proteins↗
PubMed2003

N-methyl-D-glucamine improves the laser-induced fluorescence capillary electrophoresis performance in the total plasma thiols measurement.

We describe an ultrarapid capillary electrophoresis with laser-induced fluorescence (CE-LIF) method for total plasma thiols measurement. Reduced thiols by 10% tri-n-butylphosphine (TBP) were derivatized in 10 min at room temperature with 5-iodoacetamidofluorescein (5-IAF) as fluorescent reagent. We show that CE-LIF allows a baseline separation of total plasma cysteinylglycine, homocysteine, cysteine, and glutathione in less than 5 min when N-methyl-D-glucamine in run buffer was added. CE was compared with high-performance liquid chromatography (HPLC) with fluorescence detection. The Bland-Altman test and Passing-Bablok regression demonstrates that the results obtained by CE-LIF and by HPLC are highly comparable. The simplified procedure of sample preparation, the short incubation and fast separation times, the high specificity, sensitivity and reproducibility, and the lower cost of analysis suggest that our proposed method can be considered valuable for the automation analysis in a routine laboratory.

Adult↗
PubMed2003

Association between the HFE mutations and longevity: a study in Sardinian population.

Hereditary hemochromatosis is an HLA-linked inherited disease characterised by inappropriately high absorption of iron by the gastrointestinal mucosa. The cysteine-to-tyrosine substitution at codon 282 of the HFE encoding gene sequence is responsible for the disease, although other variants, as H63D and S65C, may modify the affinity of the protein for transferrin receptors. We have recently reported that C282Y mutation is significantly increased in very old (>90 years) Sicilian women, suggesting a role in attainment of longevity. In addition, an increase of H63D polymorphism was also observed in these women but the difference was not significant. To validate and extend these results we investigated the distribution of these three common HFE gene mutations in Sardinian centenarians and controls. DNA samples, obtained from 61 controls and 57 Centenarians, were typed for HFE polymorphisms using sequence specific primers. Among the controls, none was heterozygous for the C282Y mutation, 15 were heterozygous for H63D mutation and one for S65C. Among the centenarians, none was heterozygous for the C282Y mutation whereas 25 were heterozygous for H63D mutation and four for the S65C mutation. No significant differences were observed in frequencies of the different alleles between young and centenarians both on the whole and when the data were analysed according to gender. However, there was a trend for an increased frequency of H63D allele in centenarian women (24 vs. 17%, i.e. 19/80 vs. 13/78). It is noteworthy that the cumulative frequency of H63D mutations in Centenarian and very old women from Sardinia and Sicily is 22 vs. 11%, i.e. 30/136 vs. 23/210, P=0.008. These findings are consistent with the hypothesis that there may be a survival difference for centenarian women, among carriers and non-carriers of alleles involved in iron sparing.

Aged↗
PubMed2003

Association between the HLA-DR alleles and longevity: a study in Sardinian population.

Human longevity may be correlated with optimal functioning of the immune system, suggesting that genetic determinants of longevity also resides in those polymorphisms for the immune system genes that regulate immune responses as histocompatibility (HLA) antigens. However, conflicting results have been obtained. Some well planned and designed association studies performed in Caucasians suggest that longevity is associated with positive selection of alleles (i.e. HLA-DR11) or haplotypes (i.e. HLA-B8,DR3) that confer resistance to infectious diseases, respectively, via peptide presentation or via antigen non-specific control of immune response. Association studies are subjected to a number of possible confounding factors, the homogeneity of the population in term of geographical origin among others. Because of the lack of large-scale heterogeneity, the Sardinians represent a suitable population for association studies addressed to dissect the complex traits as longevity. Thus, we have evaluated, by the amplification refractory mutation system/polymerase chain reaction, HLA-DR frequencies in 120 centenarians (79 women and 41 men) and 86 controls (53 women and 33 men) from Sardinia, to validate, in this very homogeneous population, the associations between HLA alleles or haplotypes and longevity observed in other Caucasoid populations. No significant differences were obtained by analysing the differences between Centenarians and controls except for HLA-DRB1*15 that was increased in centenarians. However, the significance was not maintained by multiplying P values for the number of alleles under study. Thus, in Sardinian centenarians, we were not able to confirm the findings observed in the well planned and designed studies performed in other Caucasoid populations. Besides, HLA HFE gene polymorphisms have been recently demonstrated to be associated with longevity in the Sicilian population but not in Danish one. On the whole these findings clearly show that HLA/longevity associations are population-specific, being heavily affected by the population-specific genetic and environmental history. So, in our opinion, HLA genes might be considered survival genes not longevity genes.

Aged↗
PubMed2002

Autosomal recessive hypercholesterolaemia in Sardinia, Italy, and mutations in ARH: a clinical and molecular genetic analysis.

BACKGROUND: Autosomal recessive hypercholesterolaemia (ARH) is caused by mutations in a putative adaptor protein called ARH. This recessive disorder, characterised by severe hypercholesterolaemia, xanthomatosis, and premature coronary artery disease, is rare except on the island of Sardinia, Italy. Our aim was to ascertain why ARH is more common on Sardinia than elsewhere. METHODS: We obtained detailed medical histories, did physical examinations, measured concentrations of lipoproteins, and harvested genomic DNA from 28 Sardinians with ARH from 17 unrelated families. We sequenced the coding regions and consensus splice sites of ARH in probands from these families, and from 40 individuals of non-Sardinian origin who had an autosomal recessive form of hypercholesterolaemia of unknown cause. FINDINGS: Two ARH mutations, a frameshift mutation (c432insA) in exon 4 (ARH1) and a nonsense mutation (c65G-->A) in exon 1 (ARH2), were present in all of the 17 unrelated families with ARH. Three of the ARH alleles contained both mutations, as a result of an ancient recombination between ARH1 and ARH2. No regional clustering of the three mutant alleles within Sardinia was apparent. Furthermore, four Italians from the mainland with autosomal recessive hypercholesterolaemia were homozygous for ARH1. INTERPRETATION: The small number, high frequency, and dispersed distribution of ARH mutations on Sardinia are consistent with these mutations being ancient and maintained in the Sardinian population because of geographic isolation.

Adolescent↗
PubMed2002

Ultrarapid capillary electrophoresis method for the determination of reduced and oxidized glutathione in red blood cells.

We describe a very rapid high-performance capillary electrophoresis method for the separation and quantification of reduced (GSH) and oxidized (GSSG) glutathione in red blood cells. Two procedures for sample preparation have been compared, Microcon-10 membrane filtration and acid precipitation. The separation is obtained in an uncoated capillary using a high ionic strength borate buffer at pH 7.8. The intra-assay coefficients of variation (CVs%) are 1.53 and 1.66 for GSH and GSSG, respectively. The run is shorter than 90 s and the migration time is highly reproducible both for GSH (CV% 0.22) and GSSG (CV% 0.17). When the filtration step is used only GSH is found, whereas both GSH and GSSG are detectable after acid precipitation, suggesting that GSSG revealed after acid treatment may be an artefact due to GSH oxidation. Because of its good analytical performance this method could be used for routine red blood cell glutathione measurement in healthy or pathological conditions.

Electrophoresis, Capillary↗
PubMedDate not supplied

Frequency of the HFE gene mutations in five Italian populations.

Genetic hemochromatosis is an autosomal recessive disorder characterized by iron overload and a variety of clinical manifestations such as liver cirrhosis and arthropathy. It is the most common genetic disease of northern European populations. The principal gene responsible for hereditary hemochromatosis, designated HFE, is located on chromosome 6 in the HLA region. The single point mutation 845A, changing cysteine at position 282 to tyrosine (C282Y), in this gene has been identified as the main genetic basis of hereditary hemochromatosis. Two other mutations, 187G, a histidine to aspartate at amino acid 63 (H63D), and 193T, a serine to cysteine at amino acid 65 (S65C), appear to be associated with milder forms of hereditary hemochromatosis. There is a high prevalence of the C282Y mutation in northern European populations, whereas in those of the Mediterranean basin the prevalence seems low and almost absent in Far East countries. This mutation seems usually to occur on the ancestral haplotype 7.1. Accordingly, a Celtic origin of this mutation has been suggested. The aim of this study was to determine the frequency of HFE gene mutations in five geographic regions in Italy. Samples were tested for C282Y, H63D, and S65C mutations of the HFE gene according to methods of each laboratory and the results were standardized with the exchange of typed samples between the different laboratories. In addition, C282Y-positive DNA samples were typed for D6S105 allele 8 and HLA-A3 by ARMS-PCR. We have found that the allele frequency of the C282Y mutation decreases from northeast Italy (Friuli, 6%) to northwest Italy (Piedmont, 4.8%) and to central Italy (Emilia-Romagna, 1.7%). However, this mutation is lacking in the two regions of the Mediterranean basin's center (Sicily and Sardinia). Accordingly, a significant difference in the frequency of the mutation was observed between these Italian regions (P = 0.07 x 10(-3)). In contrast, no difference was observed in allele frequency of H63D in the five Italian regions. Finally, as regards the S65C mutation a very low frequency was observed in Friuli, Emilia-Romagna, and Sardinia, whereas in Sicily and Piedmont we have not found this mutation. In conclusion, these data are consistent with the hypothesis that the C282Y mutation occurred in Caucasian populations of Celtic origin, whereas the H63D mutation is more ancient as demonstrated by the ubiquitous distribution.

Amino Acid Substitution↗