Short-Term Effects of Supplemental L-Arginine, Diosmin, Troxerutin, and Hesperidin in Diabetic Patients: A Pilot Study.

BACKGROUND AND AIMS: Inflammatory, oxidative stress, and endothelial dysfunction play a key role in the pathogenesis of long-term cardiovascular complications in patients with diabetes. The present observational prospective study is aimed at evaluating the effects of micronutrients and phytochemicals contained in the dietary supplement Flebotrofine® (AMNOL Chimica Biologica) on biochemical markers of inflammation, endothelial dysfunction, and glycemic control in patients with diabetes. METHODS: 105 type 1 or type 2 diabetes patients regularly took a daily dose of the dietary supplement Flebotrofine® for three consecutive months, and haematological and biochemical parameters were checked at baseline, after three months of treatment, and one month after its suspension. Statistical comparison of the laboratory parameters was performed using the two-tailed ANOVA test for repeated samples with a statistical significance level set at p < 0.05. RESULTS: The daily use of Flebotrofine® did not change the glycemic metabolic compensation of enrolled patients. After three months of regular Flebotrofine® intake, the plasma levels of the antioxidant ß-carotene and of arginine were significantly higher compared with the baseline values, with a decrease in the ADMA/arginine ratio. In contrast, apolipoprotein B, ApoB/ApoA1 ratio, and platelet and leukocyte counts significantly dropped. CONCLUSION: The daily use of Flebotrofine® might be a valid supplement of arginine, the precursor of NO, and essential in the prevention of endothelial dysfunction. The regular intake of arginine and phytochemicals also improved the antioxidant and antithrombotic profile of enrolled patients. Therefore, Flebotrofine® could be a useful dietary supplement to prevent long-term complications in patients with diabetes.

Vitamin D Supplementation Modulates ICOS+ and ICOS- Regulatory T Cell in Siblings of Children With Type 1 Diabetes.

OBJECTIVES: Vitamin D plays an immunoregulatory activity. The aim of this study was to assess the correlation between blood serum 25(OH)D levels and Th17 and Treg circulating subsets, mainly Treg/inducible costimulatory-positive (ICOS+), which seems to have a protective role in autoimmunity, in children with type 1 diabetes mellitus (T1D) and their healthy siblings (S). The secondary aim was to evaluate the impact of vitamin D supplementation on these subsets. PATIENTS AND METHODS: 22 T1D and 33 S were enrolled. Glucose, hemoglobin A1c, 25 OH vitamin D (25[OH]D), T helper type 17 (Th17; CD4+CCR6+), regulatory T cells (Treg; CD4+CD25+Foxp3+), and Treg/ICOS+ cells were evaluated. According to human leukocyte antigen (HLA) haplotypes, subjects were classified as "at risk" (HLA+), "protective haplotypes" (HLA-; "nested controls"), and "undetermined" (HLAUND). T1D and S subjects were supplemented with cholecalciferol 1000 IU/die and evaluated after 6 months. RESULTS: Vitamin D insufficiency (74.4%) and deficiency (43%) were frequent. S subjects with 25(OH)D levels <25 nmol/L had Th17, Treg (p < 0.01), and Treg/ICOS+ (P < 0.05) percentages higher than subjects with 25(OH)D >75 nmol/L. Treg/ICOS+ percentages (P < 0.05) were higher in HLA- S subjects compared to percentages observed in S with T1D. At baseline, in S subjects, a decreasing trend in Th17 and Treg/ICOS+ values (P < 0.05) from vitamin D deficiency to sufficiency was observed; 25(OH)D levels were negative predictors of Treg/ICOS+ (R2 = 0.301) and Th17 percentages (R2 = 0.138). After 6 months, supplemented S subjects showed higher 25(OH)D levels (P < 0.0001), and lower Th17 (P < 0.0001) and Treg/ICOS+ (P < 0.05) percentages than at baseline; supplemented T1D patients only had a decrease in Th17 levels (P < 0.05). CONCLUSION: Serum 25(OH)D levels seem to affect Th17 and Treg cell subsets in S subjects, consistent with its immunomodulating role. HLA role should be investigated in a larger population.

Impact of high-dose statins on vitamin D levels and platelet function in patients with coronary artery disease.

BACKGROUND: Statins represent a pivotal treatment in coronary artery disease, offering a reduction in cardiovascular risk even beyond their lipid-lowering action. However, the mechanism of these "pleiotropic" benefits of statins is poorly understood. Vitamin D has been suggested as a potential mediator of the anti-inflammatory, anti-thrombotic and vascular protecting effects of statins. Aim of present study was to assess the impact of a high-intensity statin therapy on vitamin D levels and platelet function in patients with coronary artery disease. METHODS: Patients discharged on dual antiplatelet therapy and high-intensity statins after an ACS or elective PCI were scheduled for main chemistry and vitamin D levels assessment at 30-90days post-discharge. Vitamin D (25-OHD) dosing was performed by chemiluminescence method through the LIAISON® Vitamin D assay (Diasorin Inc). Platelet function was assessed by Multiplate® (multiple platelet function analyser; Roche Diagnostics AG). RESULTS: Among 246 patients included, 142 were discharged on a new statin therapy or with an increase in previous dose (Inc-S), while 104 were already receiving a high-dose statin at admission, that remained unchanged (Eq-S). Median follow-up was 75.5days. Patients in the Inc-S group were younger (p=0.01), smokers (p<0.001), with a less frequent history of hypercholesterolemia (p=0.05), diabetes (p=0.03), hypertension (p=0.02), or previous cardiovascular events (p<0.001). They were more often admitted for an acute coronary syndrome (p<0.001) and used less anti-hypertensive drugs or nitrates. Higher total circulating calcium was observed in the Inc-S group (p=0.004), while baseline vitamin D levels were similar in the 2 groups (p=0.30). A significant reduction in the circulating low-density lipoprotein (LDL) cholesterol was observed in the Inc-S group. Vitamin D levels increased in the Inc-S patients but not in the Eq-S group (delta-25OHD: 23.2±20.5% vs 3.1±4.7%, p=0.003), with a linear relationship between the magnitude of vitamin D elevation and the reduction of LDL cholesterol (r=-0.17, p=0.01). Platelet reactivity was significantly lower in the Inc-S patients, when evaluating aggregation with different platelet activating stimuli (arachidonic acid, p=0.02, collagen, p=0.004, thrombin-activating peptide, p=0.07, ADP, p=0.002). CONCLUSIONS: In patients with coronary artery disease, the addition of a high-intensity statin treatment, besides the lipid-lowering effects, is associated to a significant increase in vitamin D levels and lower platelet reactivity, potentially providing explanation of the "pleiotropic" benefits of statins therapy in cardiovascular disease.

Vitamin D Deficiency and Glycemic Status in Children and Adolescents with Type 1 Diabetes Mellitus.

BACKGROUND: Vitamin D (25OHD) effects on glycemic control are unclear in children and adolescents with type 1 diabetes. Aims of this study were to investigate 25OHD status among children with T1DM and its relationship with insulin sensitivity and glycemic status. SUBJECTS AND METHODS: A cross sectional study was carried out between 2008-2014. A total of 141 patients had a T1DM >12 months diagnosis and were enrolled in the present study. Of these 35 (24.8%) were migrants and 106 (75.2%) Italians (T2). We retrospectively analyzed data at the onset of the disease (T0)(64 subjects) and 12-24 months before the last visit (T1,124 subjects). Fasting glucose, glycated hemoglobin (HbA1c), 25OHD levels and daily insulin requirement were evaluated and Cholecalciferol 1000 IU/day supplementation for the management of vitamin D insufficiency (<75 nmol/L) was systematically added. RESULTS: A generalized 25OHD insufficiency was found at each study time, particularly in migrants. At T0, the 25OHD levels were inversely related to diabetic keto-acidosis (DKA) severity (p<0.05). At T1 and T2, subjects with 25OHD ≤25nmol/L (10 ng/mL) showed higher daily insulin requirement (p<0.05) and HbA1c values (p<0.01) than others vitamin D status. The 25OHD levels were negatively related with HbA1c (p<0.001) and daily insulin dose (p<0.05) during follow up. There was a significant difference in 25OHD (p<0.01) between subjects with different metabolic control (HbA1c <7.5%,7.5-8%,>8%), both at T1 and T2. In supplemented subjects, we found a significant increase in 25OHD levels (p<0.0001) and decrease of HbA1c (p<0.001) between T1 and T2, but this was not significant in the migrants subgroup. Multivariate regression analysis showed a link between HbA1c and 25OHD levels (p<0.001). CONCLUSIONS: Children with T1DM show a generalized 25OHD deficiency that impact on metabolic status and glycemic homeostasis. Vitamin D supplementation improves glycemic control and should be considered as an additional therapy.

Vitamin D levels and high-residual platelet reactivity in patients receiving dual antiplatelet therapy with clopidogrel or ticagrelor.

BACKGROUND: Suboptimal platelet inhibition still represents an important challenge, especially for patients undergoing percutaneous coronary interventions (PCIs). However, very few are known so far on the predictors of high-residual platelet reactivity (HRPR) despite antiplatelet strategies. Increasing attention has been paid in the last years to the role of vitamin D in atherothrombosis. Therefore, the aim of our study was to evaluate the impact of vitamin D levels on platelet function in patients treated with dual antiplatelet therapy (DAPT). Patients treated with DAPT (ASA and clopidogrel or ticagrelor) after a recent acute coronary syndrome (ACS) or elective PCI were scheduled for platelet function assessment at 30-90 days post-discharge. Platelet function was assessed by whole blood impedance aggregometry (Multiplate®-Roche Diagnostics AG), HRPR was considered for ASPI test values > 862 AU*min (for ASA) and adenosine diphosphate (ADP) test values ≥417 AU*min (for ADP-antagonists). Fasting samples were obtained for main chemistry parameters and vitamin D level assessment. Our population is represented by 503 patients, who were divided according to vitamin D quartiles (≤9.1; 9.2-14.4; 14.5-21.7; >21.7 ng/ml). Lower vitamin D levels related with age (p = 0.04), diabetic status (p = 0.05), and previous coronary surgery (p = 0.007), therapy with beta-blockers and statins (p = 0.01 and p = 0.02). Vitamin D inversely related to the levels of total cholesterol (p = 0.01), triglycerides (p < 0.001), hemoglobin (p = 0.05), and HbA1c (p < 0.001). Significantly higher platelet reactivity was observed after platelet stimulation with ADP (p = 0.01), but not with other platelet activators. The prevalence of HRPR for ASA was low (1.2%) and not conditioned by Vitamin D levels (adjusted OR[95%CI] = 1.56[0.71-3.5], p = 0.27). HRPR with ADP-antagonists was observed in 26% of patients, and the rate increased with lower vitamin D quartiles (37.3% vs 22.2% vs 24.4% vs 20.2%, p = 0.005, adjusted OR[95%CI] = 1.23[1.02-1.49], p = 0.04). An absolute increase in HRPR with lower vitamin D levels was similarly observed among patients receiving ticagrelor (adjusted OR[95% CI] = 1.40[0.95-2.06], p = 0.08), and those on clopidogrel (adjusted OR[95%CI] = 1.31[0.99-1.75], p = 0.06). Thus, lower vitamin D levels are associated with higher platelet reactivity and impaired effectiveness of ADP-antagonists, while not influencing the effectiveness of ASA. Future studies will tell whether vitamin D supplementation can reduce platelet reactivity, overcoming the phenomenon of resistance to antiplatelet agents.

High Prevalence of Vitamin D Deficiency in Native versus Migrant Mothers and Newborns in the North of Italy: A Call to Act with a Stronger Prevention Program.

BACKGROUND: Vitamin D status during pregnancy is related to neonatal vitamin D status. Vitamin D deficiency has been associated with an increased risk of rickets in children and osteomalacia in adults. Aim of this study was to investigate 25OHD levels in maternal serum and in neonatal blood spots in native and migrant populations living in Novara (North Italy, 45°N latitude). METHODS AND FINDINGS: We carried out a cross sectional study from April 1st 2012 to March 30th 2013, in a tertiary Care Center. Maternal blood samples after delivery and newborns' blood spots were analyzed for 25OHD levels in 533 pairs. Maternal country of origin, skin phototype, vitamin D dietary intake and supplementation during pregnancy were recorded. Multivariate regression analysis, showed a link between neonatal and maternal 25OHD levels (R-square:0.664). Severely deficient 25OHD values (<25 nmol/L) were found in 38% of Italian and in 76.2% of migrant's newborns (p <0.0001), and in 18% of Italian and 48,4% of migrant mothers (p <0.0001) while 25OHD deficiency (≥25 and <50 nmol/L) was shown in 40.1% of Italian and 21.7% of migrant's newborns (p <0.0001), and in 43.6% of Italian and 41.3% of migrant mothers (p <0.0001). Italian newborns and mothers had higher 25OHD levels (34.4±19.2 and 44.9±21.2 nmol/L) than migrants (17.7±13.7 and 29.7±16.5 nmol/L; p<0.0001). A linear decrease of 25OHD levels was found with increasing skin pigmentation (phototype I 42.1 ±18.2 vs phototype VI 17.9±10.1 nmol/l; p<0.0001). Vitamin D supplementation resulted in higher 25OHD values both in mothers and in their newborns (p<0.0001). CONCLUSIONS: Vitamin D insufficiency in pregnancy and in newborns is frequent especially among migrants. A prevention program in Piedmont should urgently be considered and people identified as being at risk should be closely monitored. Vitamin D supplementation should be taken into account when considering a preventative health care policy.

Analysis of electrical and mechanical left atrial properties in patients with persistent atrial fibrillation.

The study of left atrial (LA) mechanical function during atrial fibrillation (AFib) can provide valuable information, particularly if such profiling is related to the cavity electrical substrate and conveys prognostic information. To assess if there is any relation between LA mechanical and electrical asynchrony and if such evaluation can be of interest in stratifying AFib patients. 50 patients with persistent AFib who underwent electrical cardioversion (CV) were evaluated with pre-CV atrial electrograms (AEGs). Electrical asynchrony was classified according to Wells' patterns of AEGs, ranging from most organized (I) to most dispersed (III) one. LA mechanical asynchrony was addressed by transthoracic 2D-speckle-tracking echo and quantified according to time-to-peak standard deviation (TP-SD) of wall strains and their peak values (PS) before CV, after 24 h, at 1 month. Pre-CV a linear, inverse relation between TP-SD and PS (p < 0.001) and a direct linear relation between TP-SD and Wells' classes (p = 0.04) were observed. With sinus rhythm TP-SD decreased (p = 0.023) and PS increased (p < 0.001), suggesting improved LA mechanical milieu. A multivariate analysis, testing the effects of baseline variables in predicting post-CV recurrence of AFib, revealed that amount of TP-SD variation pre/24 h post-CV was the only independent predictor at 6 months (p = 0.046). Speckle tracking-derived LA parameters can describe LA wall forces during AFib, categorizing the asynchronous mechanistic profile of AFib that correlates with the degree of the dispersed LA electrical activity. The amount of changes in LA mechanical asynchrony pre/post-CV seems to have prognostic relevance in predicting SR maintenance.

Unsuppressed parathyroid hormone in patients with autoimmune/inflammatory rheumatic diseases: implications for vitamin D supplementation.

OBJECTIVES: To verify if autoimmune/inflammatory rheumatic disease (ARD) patients were more refractory to PTH suppression by 25(OH) vitamin D (VITD). METHODS: Data from 105 consecutive ARD patients (including RA, PMR, spondyloarthritis and other CTDs) attending a tertiary-level immuno-rheumatology clinic and 1542 subjects tested at our central laboratory from 2008 to 2010 (controls) were collected. After exclusion of patients with renal failure, primary hyperparathyroidism and hypercalcaemia (n = 522), plasma VITD, PTH, calcium and phosphate concentrations were compared between these two groups. RESULTS: Plasma VITD concentrations were <25 nmol/l in 257 patients (severe deficit, 22.8%), ≥25 nmol/l but <75 nmol/l in 661 (mild deficit, 58.8%) and ≥ 75 nmol/l in 207 (normal, 18.4%). Despite similar median age, plasma VITD, calcium and phosphate values (P = 0.96, 0.30, 0.94, respectively), PTH was higher in ARD {73.0 [interquartile range (IQR) 54.2-93.7] pg/ml} than in controls [61.4 (46.9-80.3), P < 0.0002], also in all above-defined VITD categories (P < 0.05). Suppressed PTH was observed in 96.9% (95% CI 95.8%, 98.0%) of controls with VITD ≥ 75 nmol/l. However, PTH was increased more frequently in ARD vs controls. At multiple linear regression analysis, plasma VITD, age and the presence of an ARD (partial correlation coefficients -0.21, 0.15, 0.12, respectively, P < 0.0001) were independent predictors for increased PTH. CONCLUSIONS: Patients with ARD had, on average, an increased PTH concentration for any plasma VITD range, suggesting an impaired vitamin D metabolism. Therefore, vitamin D supplementation to ARD patients may be targeted to reach PTH suppression and not simply to obtain VITD concentrations considered optimal in other categories of patients.

Evaluation of amoxicillin plasma and tissue levels in pediatric patients undergoing tonsillectomy.

OBJECTIVE: Tonsillectomy is the most common surgery performed in the pediatric and young adult populations. Although recent guidelines based on meta-analysis suggest that perioperative chemoprophylaxis plays a role in reducing bacteraemia-related post-tonsillectomy complications, there is no evidence or agreement upon which specific antibiotic, dosage or administration route should be preferred. Since few previous studies have assessed the effectiveness of prophylaxis by direct measurement of antibiotic levels both in plasma and tissue, we designed an experimental study to quantitatively evaluate amoxicillin concentrations in children ready for tonsillectomy and compare these plasma and tissue levels with the Minimal Inhibitory Concentrations (MIC) of the bacteria more commonly involved in the upper airway infections. METHODS: Thirty-three pediatric patients under 14 years of age (median 5.0, IQR 4-7, range 3-11; M:F 18:15) with recurrent tonsillitis were treated with 3 doses (established on patient's weight) of amoxicillin-clavulanic acid given orally the day before plus a further dose 2h before tonsillectomy. Amoxicillin concentrations on both homogenated tonsillar cores and plasma were measured by HPLC-UV. Bacterial epidemiology and susceptibility were derived respectively from survey data collected by Microbiology Unit and MIC according to the National Committee for Clinical Laboratory Standards (NCCLS). RESULTS: Median plasma and tissue amoxicillin concentrations were respectively 4.7 microg/ml (IQR 2.1-8.0; min-max 0.4-14.3) and 1.1 microg/g (IQR 0.4-2.1; min-max 0.4-12.9), considerably below the selected target MIC of pathogens involved in the upper respiratory tract infections (S. aureus, H. influenzae, M. catarrhalis). 20 Children showed undetectable amoxicillin levels in one or both tonsils. Interestingly, 7 out of these patients (35%) had plasma concentrations higher than the target MIC (8 microg/ml). No patient displayed plasma concentrations under the limit of sensitivity of the method. Poor core-plasma and left-right core correlation was observed among patients, suggesting that fibrosis developed after recurrent tonsillitis may hamper antibiotic penetration. CONCLUSIONS: Based upon direct measurement of antibiotic levels in plasma and tissue, this study suggests that a revision of the oral prophylaxis in children is required in order to reduce microbial charge in the operative field and accordingly improve the recovery after tonsillectomy.

Low-density lipoprotein oxidizability in children with chronic renal failure.

BACKGROUND: In childhood, dyslipidemia and low-density lipoprotein (LDL) oxidation play an important role in the development of atherosclerosis. Alterations of these factors have been shown in adult uremic patients. METHODS: Nine children affected by chronic renal failure (CRF; urinary tract malformation, n = 8; polycystic kidney disease, n = 1) were studied to investigate the abnormalities of plasma lipoprotein concentration and composition and to assess the susceptibility of LDL to oxidation. All patients with CRF were on conservative treatment and, after informed consent, underwent the evaluation of (i) quantitative and qualitative plasma lipid profile; (ii) lipoprotein oxidation in vitro; and (iii) lipoprotein anti-oxidant content. These results were compared to those of an age-matched control group of eight healthy children. RESULTS: Total cholesterol, LDL and triglycerides were significantly higher in CRF than in the control group. The composition of lipoproteins was different in the two groups: the amount of anti-oxidant factors (alpha-, gamma-tocopherol and carotenoids) was different in CRF and normal controls children, while LDL susceptibility to oxidation was significantly higher in uremic children than in controls. CONCLUSIONS: CRF patients, already before dialysis, have a higher LDL oxidizability due to an altered lipoprotein composition and a low anti-oxidant content; therefore they have higher risk factors for atherosclerosis. On the basis of these data, supplementation with anti-oxidants might be useful in CRF children, but further studies are needed to evaluate the efficacy and safety of this therapeutic intervention.

Regulation of Ca2+ movements by cyclovirobuxine D in ECV304 endothelial cells.

In Fura-2/loaded ECV304 endothelial cells cyclovirobuxine D promoted a transient increase in cytosolic free Ca2+ originating from both an intracellular pool sensitive to the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and the extracellular space. The intracellular pool was apparently different from that mobilized by other agents acting through IP3 generation. The integrity of the plasma membrane was an absolute requirement. In cells treated with digitonin, cyclovirobuxine D did not promote any Ca2+ release from the intracellular stores even at high concentrations and in the absence or presence of thapsigargin or sodium azide, the inhibitors of the endoplasmic reticular or mitochondrial Ca2+ uptake. Furthermore, cyclovirobuxine D was effective in halting the persistent increase in cytosolic Ca2+ caused by thapsigargin, inhibiting the operation of the "capacitative" Ca2+ membrane channels as demonstrated by the decrease in the extent of both Ca2+-overshoot and Mn2+ influx. Additional effects of cyclovirobuxine D included a depolarization of plasma membrane apparently related to an enhanced influx of Na+ from the extracellular space. The results obtained indicate that cyclovirobuxine D markedly affects intracellular Ca2+ homeostasis in ECV304 endothelial cells by both promoting a discharge of intracellular pools and by interfering with the operation of store-dependent channels via plasma membrane depolarization.