Separation of aceclofenac and diclofenac in human plasma by free zone capillary electrophoresis using N-methyl-D-glucamine as an effective electrolyte additive.

Aceclofenac (A) and diclofenac (D) are effective non-steroidal anti-inflammatory drugs (NSAIDs) derived from the phenylacetic acid with pronounced antirheumatic, anti-inflammatory, analgesic and antipyretic properties. Our work proposes a new, fast-free zone capillary electrophoresis method for the simultaneous determination of aceclofenac and diclofenac in human plasma. The effect of increasing concentrations of N-methyl-D-glucamine organic base on borate run buffer was investigated. A good separation was achieved using a 40 cm x 75 microm uncoated silica capillary, 300 mmol/l sodium borate buffer, 200 mmol/l N-methyl-D-glucamine, pH 8.9, in about 3 min. Moreover, the plasma sample pre-treatment procedure was examined: acidic precipitants such as trichloroacetic acid (TCA), metaphosphoric acid (MPA), perchloric acid (PCA) or 5-sulphosalicylic acid (SSA) cause a total loss of analytes while acetonitrile allows a recovery of 97-98% of both compounds. Its simplicity and rapidity and the low analysis costs demonstrate that our method is a reliable and efficient mean for the comprehensive determination of aceclofenac and diclofenac in human plasma when pharmacokinetics studies are required.     

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.     

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.     

Effect of acute exercise on low molecular weight thiols in plasma

An important defence against free radicals is represented by plasma low molecular weight (LMW) thiols that compose a dynamic system of reduced and oxidized forms able to act as a buffer redox system. This study examined the effect of an acute graded exercise bout on LMW thiols in 16 young subjects (six sedentaries and 10 athletes). Blood analysis was performed before and immediately after the exercise and total and reduced thiols were measured in order to evaluate the thiol redox status. Findings suggested that the exercise test proposed was not enough to imbalance the redox status of all LMW thiols. However, when the redox status was evaluated for each thiol, it was evident that homocysteine (Hcy) redox status was significantly different after physical activity. In particular, we found a lower level of reduced Hcy after the exercise test both in sedentaries and in athletes. We concluded that duration and intensity of the proposed exercise were not enough to promote a reactive oxygen species production able to imbalance the redox thiols status and that the lowering of the reduced Hcy form may be due to the effect produced during the effort on the synthesis and/or removal processes of Hcy.     

HLA-C1 ligands are associated with increased susceptibility to systemic lupus erythematosus

Recently, the role of killer cell immunoglobulin-like receptor (KIR) in autoimmune diseases has received increasing attention. The present study was undertaken to determine the association of KIR genes and the human leukocytes antigen (HLA) ligands with Systemic Lupus Erythematosus (SLE) and accompanying oxidative stress. Presence or absence of 17 KIR and 5 HLA loci was performed using the polymerase chain reaction-sequence specific primer (PCR-SSP) method by case-control study. A total of 45 SLE patients, and 60 healthy controls, all of Sicilian descent, were enrolled. Plasma values of the anti-oxidant molecule Taurine were determined in all subjects by capillary electrophoresis UV detection. The carrier frequency of the KIR2DS2 gene was significantly increased in SLE patients compared to healthy controls (73.3 versus 45.0%; OR?=?3.36; 95% CI?=?1.46–7.74; p?=?.005) suggesting a role of KIR2DS2 gene in the susceptibility to disease. We also observed a strong positive association between the presence of HLA-C1 ligands group and the disease (82.2% in SLE patients versus 41.7% in controls; OR?=?6.47, 95% CI?=?2.58–16.26; p?<?.0001). Stepwise logistic regression analysis supported the effect of the HLA-C1 ligands in SLE patients (OR?=?7.06, 95% CI?=?0.07–2.19; p?=?.002), while the KIR genes were no longer significant. Interestingly, we found that SLE patients HLA-C1 positive showed significantly decreased plasma levels of antioxidant activity marker Taurine (69.38?±?28.49?μmol/L) compared to SLE patients HLA-C1 negative (108.37?±?86.09?μmol/L) (p?=?.03). In conclusion, HLA-C1 ligands group was significantly associated with an increased risk of SLE as well as an increased oxidative stress status overall in SLE patients.     

Influence of beta(0)-thalassemia on the phenotypic expression of heterozygous familial hypercholesterolemia : a study of patients with familial hypercholesterolemia from Sardinia

One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the alpha-globin gene (alpha-thalassemia trait) and that 6% to 17% are beta-thalassemia carriers. In this population, a single mutation of beta-globin gene (Q39X, beta(0) 39) accounts for >95% of beta-thalassemia cases. Because previous studies have shown that Sardinian beta-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the beta-thalassemia trait was also present in subjects with familial hypercholesterolemia (FH). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g>a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g>a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76+/-1.08 mmol/L in subjects with beta(0)-thalassemia trait and 8.25+/-1.66 mmol/L in subjects without this trait (P<0.001). This LDL-lowering effect was confirmed in an FH heterozygote of the same cluster who had beta(0)-thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of beta(0)-thalassemia trait emerged also when we pooled the data from all FH subjects with and without beta(0)-thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of beta(0)-thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B-containing lipoproteins. The observation that our FH subjects with beta(0)-thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of beta(0)-thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.     

Plasma homocysteine and cysteine levels in retinal vein occlusion

PURPOSE: To determine plasma homocysteine and cysteine levels in patients with retinal vein occlusion (RVO) and in healthy subjects and to ascertain whether there are statistically significant differences between patients and control subjects. METHODS: In this case-control study, the study group consisted of 75 consecutive patients with RVO: 33 had central retinal vein occlusion (CRVO), and 42 had branch retinal vein occlusion (BRVO). Seventy-two apparently healthy age- and sex-matched subjects served as control subjects. Homocysteine and cysteine levels were measured with a new laser-induced fluorescence capillary electrophoresis (CE-LIF) METHOD: Wilcoxon or Student's t-test was used, when appropriate, to determine differences between groups. RESULTS: There were no significant differences in median plasma homocysteine between patients with RVO and control subjects, nor were there any statistically significant differences when patients were categorized by type of vein occlusion (CRVO or BRVO). Similarly, there were no significant differences in mean plasma cysteine between patients with RVO and control subjects. However, when categorized by type of vein occlusion, mean plasma cysteine was significantly higher in CRVO patients than in control subjects (P = 0.034). CONCLUSIONS: This study failed to demonstrate an association between increased plasma homocysteine and RVO. Mean plasma cysteine was significantly higher in patients with CRVO, suggesting that hypercysteinemia may contribute to the pathogenesis of this retinal vascular disorder.