Vitamin D and 1-hour post-load plasma glucose in hypertensive patients.

BACKGROUND: A plasma glucose value ≥155 mg/dl for 1-hour post-load plasma glucose during an oral glucose tolerance test (OGTT) is able to identify subjects with normal glucose tolerance (NGT) at high-risk for type-2 diabetes and with subclinical organ damage. We designed this study to address if 25-hydroxyvitamin D [25(OH)D] circulating levels are associated with glucose tolerance status, and in particular with 1-hour post-load plasma glucose levels. METHODS: We enrolled 300 consecutive Caucasian hypertensive never-treated outpatients (160 men and 140 women, aged 52.9 ± 9.2 years). Subjects underwent OGTT and measurements of 25(OH)D and standard laboratory tests. Estimated glomerular filtration rate (e-GFR) was calculated by CKD-EPI formula and insulin sensitivity was assessed by Matsuda-index. RESULTS: Among participants, 230 were NGT, 44 had impaired glucose tolerance (IGT) and 26 had type-2 diabetes. According to 1-h post-load plasma glucose cut-off point of 155 mg/dL, we divided NGT subjects into: NGT < 155 (n = 156) and NGT > 155 mg/dL (n = 74).NGT ≥ 155 had higher significant fasting and post-load glucose and insulin, parathyroid hormone and hs-CRP levels than NGT < 155. On the contrary, Matsuda-index, e-GFR, and 25(OH)D were significantly lower in NGT ≥ 155 than NGT < 155 subjects. In the multiple regression analysis, 25(OH)D levels resulted the major determinant of 1-h post-load plasma glucose in all population and in the four groups of glucose tolerance status. In the whole population, Matsuda-index, hs-CRP and e-GFR explained another 12.2%, 6.7% and 1.7% of its variation. CONCLUSIONS: Our data demonstrate a significant and inverse relationship between 25(OH)D levels and glucose tolerance status, particularly with 1-h post-load glucose.

Properties of a putative cambialistic superoxide dismutase from the aerotolerant bacterium Streptococcus thermophilus strain LMG 18311.

The aerotolerance of the lactic - fermentative bacterium Streptococcus thermophilus is mainly based on the key antioxidant function of superoxide dismutase (StSOD). In this work, the comparison of recombinant StSOD (rStSOD) forms obtained from two different initiation triplets indicated that the enzyme from S. thermophilus strain LMG 18311 spans 201 residues. rStSOD is organised as a homodimer, even though protein aggregates are formed in concentrated solutions. The capability of binding and exchanging Fe or Mn in the active site classifies rStSOD as a putative cambialistic enzyme; the moderate preference for iron is counteracted by a 1.5-fold higher activity measured for the Mn-containing form. The enzyme is thermostable, being its half-inactivation time 10 min at 73.5°C; the energetic parameters of the heat inactivation process are regulated by the level of Mn cofactor. The effect of Mn content on the rStSOD sensitivity towards inhibitors and inactivators was also evaluated. Sodium azide acts as a weak inhibitor of rStSOD and its Mn content does not greatly affect this sensitivity. Concerning the physiological inactivator hydrogen peroxide, the Mn-enriched rStSOD displays a great resistance; a moderate sensitivity is instead observed in the presence of a low Mn content. Contrary to hydrogen peroxide, sodium peroxynitrite is a powerful inactivator, a behaviour enhanced in the Mn-enriched enzyme. All these results were compared with the corresponding data previously reported for the cambialistic SOD from the taxonomically related S. mutans. In S. thermophilus the regulation of the enzyme functions by the Mn content appears less relevant with respect to S. mutans.

Regulation of the properties of superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans by iron- and manganese-bound co-factor.

Streptococcus mutans, the main pathogen involved in the development of dental caries, is an aerotolerant microorganism. The bacterium lacks cytochromes and catalase, but possesses other antioxidant enzymes, such as superoxide dismutase (SmSOD). Previous researches suggested that SmSOD belongs to the 'cambialistic' group, functioning with Fe or Mn in the active site. A recombinant SmSOD (rSmSOD) with a His-tail has been produced and characterised. Studies on metal uptake and exchange proved that rSmSOD binds either Fe or Mn as a metal co-factor, even though with a consistent preference for Fe accommodation. The analysis of several enzyme samples with different values of the Mn/Fe ratio in the active site proved that the type of metal is crucial for the regulation of the activity of rSmSOD. Indeed, differently from the significant preference for Fe displayed by the enzyme in the binding reaction, its Mn-form was 71-fold more active compared to the Fe-form. The rSmSOD was endowed with a significant thermostability, its half-inactivation occurring after 10 min exposure at 71 or 73 degrees C, depending on the bound metal. Moreover, the enthalpic and entropic contribution to the heat inactivation process of rSmSOD were strongly regulated by the Mn content of the enzyme. The effect of typical inhibitors/inactivators has been investigated. rSmSOD was inhibited by sodium azide, and its sensitivity increased in the presence of higher Mn levels. Concerning two physiological inactivators, the enzyme displayed a different behaviour, being quite resistant to hydrogen peroxide and significantly sensitive to sodium peroxynitrite. Furthermore, the Mn co-factor had an amplifying role in the regulation of this different sensitivity. These results confirm the cambialistic nature of SmSOD and prove that its properties are regulated by the different metal content. The adaptative response of S. mutans during its aerobic exposure in the oral cavity could involve a different metal uptake by SmSOD.

Rat mitochondrial manganese superoxide dismutase: amino acid positions involved in covalent modifications, activity, and heat stability.

The role of three amino acid residues (Q143, Y34, S82) of rat mitochondrial superoxide dismutase (ratSOD2) in the enzymatic activity, thermostability, and post-translational modification of the enzyme was investigated through site-directed mutagenesis studies. Six recombinant forms of the enzyme were produced, carrying the Q143 or H143 residue with or without the Y34F or S82A replacement. All proteins bound manganese as active cofactor and were organized as homotetramers. The greatest effect on the activity (sixfold reduction) was observed in ratSOD2 forms containing the H143 variant, whereas Y34F and S82A substitutions moderately reduced the enzymatic activity compared to the Q143 form. Heat inactivation studies showed the high thermo-tolerance of ratSOD2 and allowed an evaluation of the related activation parameters of the heat inactivation process. Compared to Q143, the H143 variant was significantly less heat stable and displayed moderately lower enthalpic and entropic factors; the Y34F substitution caused a moderate reduction of heat stability, whereas the S82A replacement slightly improved the thermo-tolerance of the Q143 variant; both substitutions significantly increased enthalpic and entropic factors of heat inactivation, the greatest effect being observed with S82A substitution. All recombinant forms of ratSOD2 were glutathionylated in Escherichia coli, a feature pointing to the high reactivity of ratSOD2 toward glutathione. Moreover, the S82 position of the enzyme was phosphorylated in an in vitro system containing human mitochondrial protein extracts as source of protein kinases. These data highlight the role played by some residues in ratSOD2 and suggest a fine regulation of the enzyme occurring in vivo.

Clinical evolution of autoimmune thyroiditis in children and adolescents.

BACKGROUND: Few studies have addressed the clinical evolution of autoimmune thyroiditis (AIT) occurring in childhood and scant data are available on the role of thyroid ultrasonography. We aimed to evaluate the natural history of AIT diagnosed in children and adolescents and to assess the possible prognostic role of ultrasonography. METHODS: Retrospective case series prospectively followed up for a further 3-year period. RESULTS: A series of 23 patients with AIT, diagnosed before 18 years of age from 1994 to 2004, was further followed up from 2005 to 2007 with clinical, laboratory, and ultrasound evaluation. Hypothyroid patients were treated with levothyroxine (LT(4)), while euthyroid patients were left untreated. Patients with subclinical hypothyroidism were also evaluated 40 days after LT(4) withdrawal. At diagnosis seven patients were euthyroid, 14 with subclinical hypothyroidism, and two with overt hypothyroidism. Median follow-up was 4.7 years. At last follow-up visit, none of the seven euthyroid patients had developed hypothyroidism. Three of the 14 patients with subclinical hypothyroidism recovered a normal thyroid function while only one patient showed an increase in TSH level. By serological screening we identified three patients with other autoimmune disorders. CONCLUSIONS: In young patients with normal or mildly increased TSH levels and minimal echographic changes, AIT may remain stationary for years. In fact, patients with subclinical hypothyroidism recover a normal thyroid function in approximately 20% of cases. In patients with subclinical hypothyroidism and goiter, LT(4) therapy may induce thyroid size reduction. Screening for other autoimmune disorders is useful to identify patients that need further diagnostic assessment.

Ascaris lumbricoides-induced suppression of total and specific IgE responses in atopic subjects is interleukin 10-independent and associated with an increase of CD25(+) cells.

Ascaris presence in humans has been associated with high levels of blood eosinophils and serum IgE. This study was designed to address the influence of Ascaris infection on allergic and inflammatory parameters of atopic subjects. A cross-sectional design was used, and atopic individuals to be assessed were divided into 3 groups including Ascaris-infected, anti-Ascaris IgG-positive (seropositive), and control subjects. All subjects enrolled had positive skin test reactivity to at least 1 perennial or seasonal allergen; however, levels of C-reactive protein, C3, and C4 were within normal range values. Eosinophil percentage was not significantly different among the groups studied. Total IgE and specific anti-Ascaris IgE levels in the seropositive group were significantly higher than concentrations found in both control and infected groups. Interleukin (IL)-4 release in Ascaris-infected patients was significantly increased versus seropositives, who were able to produce more IL-4 than controls. The levels of IL-10 were lower in the seropositives as well as infected subjects in comparison with controls. CD25(+) lymphocyte populations were significantly increased in the infected group versus the seropositives as well as the controls. Lung function tests of some selected seropositive subjects were significantly impaired. The same parameters of a representative infected patient were not different from controls. Our data on T helper type 2 cells (Th2) and regulatory T cells (Treg) features, as well as CD25(+) lymphocyte increase, suggest an Ascaris-induced mechanism leading to parasite survival. Moreover, the stable control of both T helper type 1 cells (Th1) and Th2 immunity cascades, paralleled by the absence of overwhelming inflammatory systemic reactions and lack of allergic syndromes, may result in a favorable host condition.

Glutathionylation of the iron superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis.

Our previous work showed that the adduct between beta-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism.

Effects of growth hormone and insulin-like growth factor-1 on cardiac hypertrophy of hypertensive patients.

OBJECTIVES: Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) interfere with cardiac mass (left ventricular mass; LVM) development. We investigated the role of the GH/IGF-1 axis on LVM and ventricular geometry in a group of 230 never-treated hypertensive patients. METHODS: Partition values for left ventricular hypertrophy (LVH) were 125 g/m2 for both women and men. Insulin resistance was estimated by the homeostasis model assessment (HOMA) index. RESULTS: A significant inverse correlation was observed between IGF-1 and both fasting insulin (r = -0.249; P < 0.0001) and GH (r = -0.218; P < 0.0001). Systolic blood pressure (157.3 +/- 13.6 versus 149.4 +/- 12.8 mmHg; P < 0.001), fasting insulin (17.4 +/- 8.5 versus 11.4 +/- 6.0 microU/l; P < 0.0001), HOMA (4.4 +/- 2.3 versus 2.9 +/- 1.6; P < 0.0001) and GH (1.0 +/- 1.0 versus 0.4 +/- 0.5 ng/ml; P < 0.0001) were significantly higher in patients with LVH; on the contrary, IGF-1 values (119.1 +/- 47.8 versus 160.1 +/- 75.5 ng/ml; P < 0.0001) were higher in patients without LVH. In a logistic regression analysis, the strongest independent predictors of LVH were GH [relative risk (RR) = 2.078; 95% confidence interval (CI) = 1.364-3.163], HOMA (RR = 1.345; 95% CI = 1.133-1.596), IGF-1 (RR = 0.993; 95% CI = 0.998-0.999) and systolic blood pressure (RR = 1.036; 95% CI = 1.013-1.060). IGF-1 showed an opposite trend in patients with eccentric and concentric hypertrophy. CONCLUSIONS: Present data demonstrate that the increase in LVM prevalent in human essential hypertension is directly associated with serum GH levels and inversely related to circulating IGF-1.

Detection and functional analysis of an SNP in the promoter of the human ferritin H gene that modulates the gene expression.

The H ferritin promoter spans approximately 150 bp, upstream of the transcription start and is composed by two cis-elements in position -132 (A box) and -62 (B-box), respectively. The A box is recognized by the transcription factor Sp1, and the B-box by a protein complex called Bbf, which includes the CAAT binding factor NF-Y. In this study we performed a functional analysis of an H ferritin promoter allele carrying a G to T substitution adjacent to the Bbf binding site, in position -69. In vitro studies with reporter constructs revealed a significantly reduced transcriptional activity of this allele compared to that of the w.t. promoter that was mirrored by a decrease in Bbf binding. In vivo, this variant genotype is accompanied by a reduced amount of the H mRNA in peripheral blood lymphocytes.

Risk factors for congenital hypothyroidism: results of a population case-control study (1997-2003).

OBJECTIVE: To identify risk factors for permanent and transient congenital hypothyroidism (CH). DESIGN: A population-based case-control study was carried out by using the network created in Italy for the National Register of Infants with CH. METHODS: Four controls were enrolled for each new CH infant; 173 cases and 690 controls were enrolled in 4 years. In order to distinguish among risk factors for permanent and transient CH, diagnosis was re-evaluated 3 years after enrollment when there was a suspicion of transient CH being present. Familial, maternal, neonatal and environmental influences were investigated. RESULTS: An increased risk for permanent CH was detected in twins by a multivariate analysis (odds ratio (OR) = 12.2, 95% confidence interval (CI): 2.4-62.3). A statistically significant association with additional birth defects, female gender and gestational age >40 weeks was also confirmed. Although not significant, an increased risk of CH was observed among infants with a family history of thyroid diseases among parents (OR = 1.9, 95% CI: 0.7-5.2). Maternal diabetes was also found to be slightly associated with permanent CH (OR = 15.7, 95% CI: 0.9-523) in infants who were large for gestational age. With regard to transient CH, intrauterine growth retardation and preterm delivery were independent risk factors for this form of CH. CONCLUSION: This study showed that many risk factors contribute to the aetiology of CH. In particular, our results suggested a multifactorial origin of CH in which genetic and environmental factors play a role in the development of the disease.

EDTA is essential to recover lead from dried blood spots on filter paper.

BACKGROUND: Residual dried blood spots (DBSs) on filter paper from neonatal screening have been proposed as samples for population survey of lead contamination. We have investigated the EDTA effect on lead release in the eluting solution. METHODS: Furnace atomic absorption spectrophotometry has been used for lead measurements. Standard, blank and sample solutions contained 2% m/v NH(4)H(2)PO(4), 0.5% v/v Triton X-100 and 0.2% v/v HNO(3) as matrix modifier solution (MMS) with or without EDTA. A calibration curve was established from aqueous standard solutions. Paper discs from DBS and blank, punched near the DBS, were eluted in MM solution and, where required, EDTA at different concentrations. Specimens were leftover DBSs with different storage times, matched samples from 20 adult patients consisting of liquid whole blood (LWB) containing 5 mmol/L EDTA, DBSs eluted in MM solution with 5 mmol/L EDTA or without EDTA. RESULTS: Optimal lead recovery from DBS required 5 mM EDTA in the eluting solution. Mean lead levels of LWB and DBSs eluted with EDTA were similar and higher than DBSs without EDTA (P<0.001). Without EDTA, the median value of lead optical density was lower for 6-month-old DBSs than for blanks (P<0.001). CONCLUSIONS: Residual DBSs can be used for population survey, but 5 mmol/L EDTA in the extracting solution is required to fully recover lead.