Biomarkers of cell damage, neutrophil and macrophage activation associated with in-hospital mortality in geriatric COVID-19 patients.

BACKGROUND: The risk for symptomatic COVID-19 requiring hospitalization is higher in the older population. The course of the disease in hospitalised older patients may show significant variation, from mild to severe illness, ultimately leading to death in the most critical cases. The analysis of circulating biomolecules involved in mechanisms of inflammation, cell damage and innate immunity could lead to identify new biomarkers of COVID-19 severity, aimed to improve the clinical management of subjects at higher risk of severe outcomes. In a cohort of COVID-19 geriatric patients (n= 156) who required hospitalization we analysed, on-admission, a series of circulating biomarkers related to neutrophil activation (neutrophil elastase, LL-37), macrophage activation (sCD163) and cell damage (nuclear cfDNA, mithocondrial cfDNA and nuclear cfDNA integrity). The above reported biomarkers were tested for their association with in-hospital mortality and with clinical, inflammatory and routine hematological parameters. Aim of the study was to unravel prognostic parameters for risk stratification of COVID-19 patients. RESULTS: Lower n-cfDNA integrity, higher neutrophil elastase and higher sCD163 levels were significantly associated with an increased risk of in-hospital decease. Median (IQR) values observed in discharged vs. deceased patients were: 0.50 (0.30-0.72) vs. 0.33 (0.22-0.62) for n-cfDNA integrity; 94.0 (47.7-154.0) ng/ml vs. 115.7 (84.2-212.7) ng/ml for neutrophil elastase; 614.0 (370.0-821.0) ng/ml vs. 787.0 (560.0-1304.0) ng/ml for sCD163. The analysis of survival curves in patients stratified for tertiles of each biomarker showed that patients with n-cfDNA integrity < 0.32 or sCD163 in the range 492-811 ng/ml had higher risk of in-hospital decease than, respectively, patients with higher n-cfDNA integrity or lower sCD163. These associations were further confirmed in multivariate models adjusted for age, sex and outcome-related clinical variables. In these models also high levels of neutrophil elastase (>150 ng/ml) appeared to be independent predictor of in-hospital death. An additional analysis of neutrophil elastase in patients stratified for n-cfDNA integrity levels was conducted to better describe the association of the studied parameters with the outcome. CONCLUSIONS: On the whole, biomarkers of cell-free DNA integrity, neutrophil and macrophage activation might provide a valuable contribution to identify geriatric patients with high risk of COVID-19 in-hospital mortality.

Randomized, double-blind, placebo-controlled trial to evaluate the effect of Helicobacter pylori eradication on glucose homeostasis in type 2 diabetic patients.

BACKGROUND AND AIMS: Literature data suggest an association between Helicobacter pylori infection and glucose homeostasis. However, a causative link between them has not been demonstrated yet. The aim of this study is to investigate the effect of H. pylori eradication on glucose homeostasis in patients with type 2 diabetes. METHODS AND RESULTS: A randomized, double-blind, placebo-controlled trial was conducted to investigate the effect of H. pylori eradication on glucose homeostasis in 154 patients with type 2 diabetes and who tested positive for H. pylori infection (mean age (SD), 63.1 (8.1) years). Subjects were assigned to H. pylori eradication treatment or placebo. Metabolic and inflammatory parameters were measured in all subjects at baseline and 4 weeks after the treatment. H. pylori eradication led to an improvement in glucose homeostasis, measured by HOMA-IR (p < 0.001) and KITT (0 = 0.041), due to the decrease in fasting insulin levels (p = 0.004). The results also showed that lower levels of inflammatory parameters were present after eradication. CONCLUSION: To our knowledge this is the first randomized, double blind, controlled study where the effect of H. pylori eradication on glucose homeostasis in subjects with type 2 diabetes has been investigated. Our findings demonstrate that H. pylori eradication improves glucose homeostasis in patients with type 2 diabetes through a decrease in pro-inflammatory factors. TRIAL REGISTRATION NUMBER: ACTRN12609000255280 (https://www.anzctr.org.au/).

Diabetes-related quality of life is enhanced by glycaemic improvement in older people.

AIMS: To investigate the validity and reliability of the Audit of Diabetes-Dependent Quality of Life instrument in older Italians with diabetes and to test the association of diabetes-related quality of life with glycaemic control over time. METHODS: A total of 558 outpatients with Type 2 diabetes from the Diabetic Unit of the Italian National Research Centre on Aging Hospital in Ancona were enrolled to complete questionnaires (Audit of Diabetes-Dependent Quality of Life-19 and the Short-Form-12), and to undergo clinical and biochemical testing at baseline and at 12 months of follow-up. The overall impact of diabetes using the average weighted impact score from the Audit of Diabetes-Dependent Quality of Life questionnaire was calculated. Participants were categorized according to this score as having either less or more negative diabetes-related quality of life. RESULTS: Participants had a mean ± SD age of 67.7 ± 9.2 years and 51.8% were male. Factor analysis and Cronbach's coefficient of internal consistency (Cronbach's α = 0.931) confirmed that the 19 domain-specific Audit of Diabetes-Dependent Quality of Life items could be combined into a single scale in this Italian population. The impact score correlated with the physical (r = 0.275; P < 0.001) and mental components (r = 0.291; P < 0.001) of the Short-Form-12 questionnaire. Significant differences were found according to diabetic complications in specific Audit of Diabetes-Dependent Quality of Life items and impact scores. Insulin use had a greater association with a more negative quality of life compared with other antidiabetic agents. A multivariate linear regression model with restricted linear spline application showed that the relationship between HbA1c and impact score was not linear and that the change in the impact score was associated with improved glycaemic control in those with a less negative diabetes-related quality of life at 12 months. CONCLUSIONS: The Audit of Diabetes-Dependent Quality of Life-19 is a valid tool for measuring the impact of diabetes on quality of life in older Italians. Perception of diabetes-related quality of life is associated with glycaemic control over time.

Hyperglycemia following recovery from hypoglycemia worsens endothelial damage and thrombosis activation in type 1 diabetes and in healthy controls.

BACKGROUND AND AIMS: Hypoglycemia produces thrombosis activation, but little attention has been paid to the effects of hyperglycemia following recovery from hypoglycemia on thrombosis activation. METHODS AND RESULTS: In both twenty-two healthy subjects and twenty-one matched persons with type 1 diabetes, recovery from a 2-h induced hypoglycemia was obtained by reaching normo-glycemia or hyperglycemia for another 2 h. After this, normal glycemia was maintained for the following 6 h. Hyperglycemia after hypoglycemia was also repeated with the concomitant infusion of vitamin C. In both controls and people with diabetes, the recovery with normo-glycemia was accompanied by a significant improvement of Von Willebrand factor (vWF), prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin III-complexes (TAT), P-selectin, plasminogen activator inhibitor-1 (PAI-1), nitrotyrosine and 8-iso-prostaglandin F2α (8-iso-PGF2α) (p < 0.01 vs hypoglycemia for all the parameters), all directly affected by hypoglycemia itself (p < 0.01 vs baseline for all the parameters). On the contrary, the recovery with hyperglycemia after hypoglycemia worsens all these parameters (p < 0.01 vs normoglycemia for all the parameters), an effect persisting even after the additional 6 h of normo-glycemia. The effect of hyperglycemia following hypoglycemia was partially counterbalanced when vitamin C was infused (p < 0.01 vs hyperglycemia alone for all the parameters), suggesting that hyperglycemia following hypoglycemia may activate thrombosis through the oxidative stress production. CONCLUSION: This study shows that, in type 1 diabetes as well as in controls, the way in which recovery from hypoglycemia takes place could play an important role in favoring the activation of thrombosis and oxidative stress, widely recognized cardiovascular risk factors.

Leukocyte telomere length is associated with complications of type 2 diabetes mellitus.

OBJECTIVE: The key goal of diabetes management is to prevent complications. While the patho-physiological mechanisms responsible for diabetes complications have been extensively studied, at present it is impossible to predict which patient with diabetes could develop complications. In recent years, the role of leukocyte telomere length in the pathogenesis of cardiovascular disease and Type 2 diabetes has been investigated. However, studies aiming to investigate the role of telomeres in the development and progression of Type 2 diabetes, as well as diabetic complications, are still lacking. As a consequence, this study aimed to verify whether leukocyte telomere length is associated with the presence and the number of diabetic complications in a sample of patients with Type 2 diabetes. METHODS: This is a cross-sectional study. Nine hundred and one subjects were enrolled, including 501 patients with Type 2 diabetes, of whom 284 had at least one complication and 217 were without complications, and 400 control subjects. Leukocyte telomere length was measured by quantitative real-time PCR. RESULTS: Patients with diabetes complications had significantly shorter leukocyte telomere length than both patients without diabetes complications and healthy control subjects. Moreover, among patients with diabetes complications, leukocyte telomere length became significantly and gradually shorter with the increasing number of diabetes complications. The magnitude of the effect of the decrease of the abundance of telomeric template vs. a single-copy gene length (T/S ratio) on complications is described by the estimated odds ratio OR=5.44 (95%CI 3.52-8.42). CONCLUSIONS: The results of the study support the hypothesis that telomere attrition may be a marker associated with the presence and the number of diabetic complications.

+647 A/C and +1245 MT1A polymorphisms in the susceptibility of diabetes mellitus and cardiovascular complications.

Diabetes mellitus is a chronic disease characterized by an overproduction of reactive oxygen species, which perturbs zinc metabolism and promotes the onset of cardiovascular disease (CVD) in diabetic patients. Metallothioneins (MT) are cysteine-rich metal-binding proteins which, by means of their antioxidant and zinc-buffering properties, might prevent the development of diabetic cardiovascular complications. A recent investigation shows that a polymorphism (+647 A/C) in the human MT-1A gene, affects the intracellular zinc ion release (iZnR) from the proteins and is associated with longevity in Italian population. The aim of the present study is to assess the involvement of +647 A/C and +1245 A/G MT1A polymorphisms with the susceptibility to type 2 diabetes (DM2) and cardiovascular complications. The study included 694 old individuals: 242 old healthy controls, 217 DM2 patients without clinical evidence of CVD (DNC) and 235 diabetic patients with diagnosis of CVD (DCVD). +647 A/C MT1A polymorphism, but not the second SNP, was associated with DM2. C allele carriers were more prevalent in DNC and DCVD patients than in control group (OR=1.37, p=0.034; OR=1.54, p=0.002, respectively). C+ carriers was associated with higher glycemia and glycosylated hemoglobin in DCVD patients, but not in DNC or control subjects. No differences in plasma zinc, but a modulation of MT levels and iZnR in PBMCs were observed in DCVD cohort when related to +647 A/C MT1A polymorphism. In summary, this work provides novel evidence on the association of the +647 A/C MT1A polymorphism with DM2. Moreover, C+ carriers in DCVD patients presented a worse glycemic control, a reduced iZnR and a higher MT levels, suggesting a possible role of MT in diabetic cardiovascular complications.

C-reactive protein is directly related to plasminogen activator inhibitor type 1 (PAI-1) levels in diabetic subjects with the 4G allele at position -675 of the PAI-1 gene.

BACKGROUND AND AIMS: C-reactive protein (CRP) has been identified as a possible factor able to promote atherosclerosis. "In vitro" studies have demonstrated that CRP induces plasminogen activator inhibitor type 1 (PAI-1) expression, suggesting a hypofibrinolytic role for CRP. As CRP and PAI-1 levels increase in type 2 diabetic subjects, we decided to study the relationship between CRP and PAI-1, and the role of the 4G/5G polymorphism of the PAI-1 gene on this relationship in a diabetic population without complications. METHODS AND RESULTS: Two hundred and ninety-five type 2 diabetic patients (age 60.9+/-10.5 years) and 290 healthy controls (age 59.2+/-11.5 years) were enrolled. A significant correlation between PAI-1 and CRP in diabetic subjects was found (r=0.45, p<0.001), whereas no relationship was evident in the control subjects between these inflammatory markers. Multiple regression analysis highlighted that CRP is the only one significant variable of PAI-1 antigen in diabetic subjects (partial r=0.31, p<0.01). Stratifying by genotype, a positive correlation between PAI-1 and CRP in 4G/4G (partial r=0.64 p<0.001) and 4G/5G (partial r=0.47, p<0.001) subjects was found, whereas no correlation in 5G/5G was present. Multiple regression analysis confirmed the presence of this correlation in 4G/4G (partial r=0.45, p<0.001) and in 4G/5G (partial r=0.34, p=0.007) diabetic patients. CONCLUSIONS: These findings demonstrate that CRP plays an important role in the complex mechanism regulating PAI-1 antigen in 4G diabetic carriers.

ZnT8 Arg325Trp polymorphism influences zinc transporter expression and cytokine production in PBMCs from patients with diabetes.

AIMS: ZnT8 Arg325Trp polymorphism has been associated with type 2 diabetes (T2DM) susceptibility. The Arg-325 risk variant shows accelerated zinc (Zn) transport kinetic and reduced glucose-stimulated insulin secretion in pancreatic cells. However, it remains unexplored the role of Znt8 polymorphism in the regulation of Zn homeostasis and inflammatory response in peripheral blood mononuclear cells (PBMCs) from T2DM patients. METHODS AND RESULTS: A total of 556 healthy controls and 413 T2DM patients were genotyped for ZnT8 Arg325Trp polymorphism confirming the association of Arg-325 variant with an increased T2DM risk (OR = 1.35 95% C.I: 1.10-1.66; p = 0.0044). Moreover, PBMCs from Arg/Arg T2DM subjects showed increased intracellular free Zn, higher gene expression of Metallothioneins, Znt1, Znt8, Zip2 genes, and reduced Znt4 and Znt7. Higher release of IL-1α, IL-1ß, IFN-γ, IL-12p70 and TNF-α and a reduced IL-10 secretion after lipopolysaccharide (LPS) stimulation were observed in PBMCs from Arg/Arg T2DM carriers as compared to subjects with the Trp variant. CONCLUSIONS: Our data provide evidence of a substantial different Zn homeostasis regulation between Znt8 Arg-325 and Trp-325 carriers in PBMCs from T2DM patients. Moreover, Znt8 Arg-325 risk variant shows an enhanced inflammatory response upon LPS stimulation that might aggravate insulin resistance and the progression of diabetes cardiovascular complications.

Effect of etanercept on insulin sensitivity in nine patients with psoriasis.

Metabolic syndrome is associated to chronic low grade inflammation, characterized by increased levels of inflammatory cytokines, such as Tumor Necrosis Factor-alpha (TNF-alpha) and Interleukin-6 (IL-6). In particular, TNF-alpha causes a decrease in the insulin-stimulated kinases related to the early phases of the insulin cascade, thereby leading to insulin resistance. Etanercept is a human fusion protein used in the treatment of psoriasis and inflammatory arthritis. It blocks inflammatory response by interfering in the binding of TNF-alpha to its receptors. The aim of this case report study is to verify the effect of Etanercept on insulin sensitivity, lipid profile and inflammatory status in psoriatic patients. Nine psoriatic patients with stable, active, plaque type psoriasis were enrolled and treated with Etanercept for 24 weeks. We found an improvement in the metabolic assessment with a significant reduction of insulin plasma levels. In particular, this treatment allows to maintain their euglycemic state with lower insulin plasma levels, as confirmed by the improved Homeostasis Model Assessment (HOMA) index. We conclude that Etanercept, probably acting on inflammation, improves insulin sensitivity in psoriatic subjects.

Interleukin-6-174 G > C polymorphism affects the association between IL-6 plasma levels and insulin resistance in type 2 diabetic patients.

Interleukin-6 (IL-6), a powerful inflammatory mediator, plays a pivotal role in the pathogenesis of insulin resistance and type 2 diabetes. Recently, the IL-6 promoter polymorphism, at position -174 (G > C), has been associated to insulin sensitivity although contrasting data have been reported. The aim of this study was to evaluate the effect of the IL-6-174 G > C polymorphism on insulin resistance. In 238 type 2 diabetic patients without diabetic complications and in 255 control subjects, age and gender-matched, we evaluated the IL-6 -174 G > C genotype, the IL-6 plasma levels and the insulin resistance by the homeostasis model assessment (HOMA). The levels of IL-6 and HOMA were not genotype-dependent and were higher in diabetic patients (p < 0.01). Control subjects, both C+ (CG + CC genotypes) and C- (GG genotype) carriers, showed IL-6 plasma levels significantly related to BMI, fasting insulin and HOMA. The same relationships were found in C+ diabetic carriers. Differently, diabetic C- carriers did not show any relationship between IL-6 levels and all the evaluated variables. Interestingly, all the correlations were dependent on BMI. These findings highlight that IL-6-174 G > C polymorphism affects insulin resistance in type 2 diabetes, where C+ carriers have an insulin resistance "IL-6-sensitive", while C- carriers do not. The identification of two categories of diabetic patients may, therefore, lead to different therapeutic strategies in the management of insulin resistance.

Fast, simple and cost-effective determination of thiopental in human plasma by a new HPLC technique.

BACKGROUND: Thiopental is an anaesthetic drug that is largely used in both short-term and long-term infusion. After long-term infusion of thiopental, non-linear and inter-individual-dependent pharmacokinetics occur because of the saturation and/or induction of the metabolism. Clinical monitoring is important so that therapeutic adjustments can be made in many of the different pharmacological treatments, especially when long-term infusion is required. We describe a new, rapid HPLC method for the determination of plasma thiopental. METHODS: Sample preparation involved precipitation of plasma proteins using a mixture of methanol, zinc sulfate and ethylene glycol, and containing the internal standard 5-ethyl-5-p-tolyl-barbituric acid. After adding trichloroacetic acid, the sample was centrifuged and the supernatant was injected into a C(18) reversed-phase column. The mobile phase used was water-methanol-acetonitrile (50:40:10, v/v). The eluent was monitored at 290 nm. RESULTS: The calibration curve was linear from 0.2 to 100 microg/mL. Precision, calculated as the coefficient of variation (%), was in the range of 3.62-0.70% for the within-day assay and 5.77-1.51% for the between-day assay. The absolute recoveries obtained from supplemented samples were never less than 100%. CONCLUSIONS: This technique shows good reliability and seems to be suitable for a very fast and simple therapeutic monitoring of plasma thiopental.

Relationship between plasminogen activator inhibitor type-1 plasma levels and the lipoprotein(a) concentrations in non-insulin-dependent diabetes mellitus.

The first part of the paper deals with the relationship between two inhibiting factors of the complex enzyme cascade regulating fibrinolysis, namely plasminogen activator inhibitor type-1 (PAI-1) and lipoprotein(a) (Lp(a)). Blood concentrations of Lp(a), PAI-1 antigen (PAI-1 AG) and activity (PAI-1 AT), and the main parameters of lipo- and glyco-metabolic balance were studied in 80 type II diabetic patients. Roughly hyperbolic patterns have been found between PAI-1 and Lp(a). Negative statistically significant linear correlation can be elicited when Log PAI-1 AG and Log PAI-1 AT values are plotted versus Lp(a) values, the first one being particularly tight. These findings suggest a nearly on/off control of the two parameters, limiting the risk of hypofibrinolysis. The second part of the paper was aimed at verifying this hypothesis. A group of 30 diabetic patients were treated for 3 months with metformin, an antidiabetic biguanide compound which has been reported to reduce PAI-1 levels both in diabetic and in non-diabetic patients. Metformin significantly reduced PAI-1 AG and PAI-1 AT but did not influence plasma Lp(a) levels. A clear linear correlation between the basal Lp(a) values and the changes in PAI-1 AG levels was found. An even tighter correlation was elicited between the decrease in PAI-1, and PAI-1 pretreatment values.

Glycosylated hemoglobin and fructosamines: does their determination really reflect the glycemic control in diabetic patients?

The present experiment was designed to determine whether scavenging capacity of serum, in addition to glucose level, influences hemoglobin and serum protein glycosylation in non-insulin dependent diabetic patients. For this purpose forty-seven patients homogeneous for age, disease duration, therapy and glyco-metabolic control were selected. Fasting and post-prandial glycemia and insulinemia as well as glycosuria were weekly analysed during the sixty days preceding glycosylated hemoglobin (HbA1c), fructosamines and serum scavenging capacity determination. This last parameter has been evaluated by a method based on the property of beta-phycoerythrin (beta-PE) to loss its fluorescence when damaged by oxygen radicals, that were produced by Cu++ and H2O2. The oxygen radical absorbance capacity (ORACOH) of serum was assayed as the ability of serum to delay the loss of beta-PE fluorescence. As expected, a statistically significant positive correlation was found comparing both fructosamines and HbA1c levels with mean fasting glycemia measured over twenty and sixty days, respectively. The key result of this study is represented by the finding that both HbAlc and fructosamines levels show a statistically significant negative correlation with ORACOH values. This correlation can explain a large percent of the data dispersion occurring when ORACOH is not taken into account. In order to better describe the role of ORACOH, patients were separated into two sub-groups with an ORACOH lower (L-ORACOH) and greater (H-ORACOH) than 100 U/ml. Examining the correlation between mean fasting glycemia and the two glycosylated proteins considered in these two sub-groups, curves with different slopes were obtained, supporting that the rate of glycosylation of both proteins was higher in L-ORACOH patients as compared to those with H-ORACOH. Present data suggest that for a proper interpretation of the HbA1c and fructosamines data in diabetic patients, the scavenging capacity level of serum should be taken into account.

Relationship between lipoprotein(a) levels, oxidative stress, and blood pressure levels in patients with essential hypertension.

High plasma levels of lipoprotein(a) [Lp(a)] are considered a risk factor for the development of coronary artery disease. In vitro experiments have shown that oxidized Lp(a) is able to impair the arterial endothelium-dependent dilation, thus suggesting a possible role of Lp(a) in the genesis of essential hypertension. The aim of our work was to investigate the correlation of blood pressure levels with plasma Lp(a) concentration, apo(a) isoform size, and peroxidative stress in patients with essential hypertension. The study was performed in 54 untreated hypertensive patients whose blood pressure was monitored for 24 h by ambulatory blood pressure monitoring. Lp(a) concentration was measured by a double monoclonal antibody-based enzyme immunoassay demonstrated to be insensitive to apo(a) size heterogeneity. Apo(a) isoforms were determined by a high-resolution SDS-agarose gel electrophoresis followed by immunoblotting. A significant correlation was found between Lp(a) levels and the night-time systolic and diastolic pressures (r=0.32, P<0.05 and r=0.30, P<0.05, respectively), as well as with the mean night-time fall in systolic and diastolic blood pressures (r=-0.28, P<0.05 and r=-0.29, P<0.05, respectively). These relationships were further potentiated when peroxidative stress data were taken into consideration (r=0.37 and r=0.40, P<0.01 for the night-time systolic and diastolic pressures, respectively and r=-0.34 and r=-0.38, P<0.01 for the night-time fall in systolic and diastolic blood pressures, respectively). Apo(a) isoform size did not affect these relationships. Our data suggest that Lp(a) and peroxidative stress may be involved as cofactors in essential hypertension, with a mechanism that remains to be elucidated.

Risk profiles in type 2 diabetes (metabolic syndrome): integration of IL-10 polymorphisms and laboratory parameters to identify vascular damages related complications.

Recently it has been reported that low serum IL-10 levels are associated with an increased susceptibility for metabolic syndrome and type 2 diabetes mellitus (T2DM). We investigated whether the -1087G/A (rs1800896), -824C/T (rs1800871), -597C/A (rs1800872) IL-10 polymorphisms were associated with type 2 diabetes in a study on a cohort of Italian Caucasians comprising 490 type 2 diabetic and 349 control subjects. Stratifying the data according to IL-10 genotypes, trends for the progressive increase of glucose and neutrophil levels were observed in -1087GG vs. -1087GA vs. -1087AA positive diabetic patients (-1087GG<-1087GA<-1087AA). In addition, evaluating the laboratory parameters according to the -597/-824/-1087 derived haplotypes a significant increase of neutrophils was found in diabetic vs. non-diabetic -597A/ -824T/-1087A positive subjects (Student t test = 3.707, p<0.01). In an attempt to integrate clinical laboratory and immunogenetic data to determine whether these factors taken together define sufficient risk sets for type 2 diabetes we performed the grade-of-membership analysis (GoM). GoM allowed to identify a population of subjects negative for IL-10 -824T allele, 74.4% of which were diabetic patients characterised by vascular damages (Chronic kidney failure and/or Myocardial Infarction), reduction of haematocrit, increase of blood urea nitrogen, creatinin and monocyte levels. These data seem to suggest that -597A/-824T/-1087A negative subjects are more prone to the major type 2 diabetic vascular damages and allow to hypothesise that the contemporary evaluation of some simple hematochemical parameters and IL-10 SNPs may allow identifying diabetic patients with the worse prognostic profile, needing both better complication prevention planning and therapeutic strategies.

Increased expression of 5-lipoxygenase is common in clear cell renal cell carcinoma.

The clinical behaviour of Clear Cell Renal Cell Carcinoma (CC-RCC) is often unpredictable. To fully understand the signaling pathways involved in CC-RCC development, we examined whether the 5-Lipoxygenase (5-LO), which catalyzes the biosynthesis of proinflammatory leukotrienes, is involved in renal tumorigenesis. By analyzing 46 snap-frozen primary renal cell carcinomas and their corresponding normal renal cortex biopsies, 5-LO protein levels were found to be significantly increased in the majority of CC-RCCs (P<0.001). Quantitative 5-LO mRNA expression analysis revealed up to 3-fold increased expression in the tumor tissues. There was no association between 5-LO and gender, grade or vein invasion. In contrast, increased 5-LO protein and mRNA correlated with large tumor size (>4 cm) and age of patients (P<0.001). 5-LO was frequently overexpressed in von Hippel-Lindau protein (pVHL)-reduced tumors and in Vascular Endothelial Growth Factor (VEGF)-positive tumors, which represent two frequent alterations in CC-RCC. Cell culture experiments demonstrated that VEGF expression was strongly inducible by 5-LO metabolites in RCC cell lines. The loss of pVHL expression led to high basal 5-LO and VEGF expression, which were markedly reduced by transfection with 5-LO small interfering RNA (siRNA). These results suggest that 5-LO up-regulation is an important step in renal cancer progression.

Biochemical characterization of density-separated trout erythrocytes.

1. A discontinuous gradient in the region of 45-65% "Percoll" has been utilized for the separation of trout erythrocytes. Three different fractions were obtained. 2. We have evaluated antioxidant enzyme activities and membrane fluidity. The results indicated that catalase and glutathione peroxidase activities increased with the density of the fraction while the membrane fluidity was unchanged. 3. The observed results show a marked difference between nucleated (fish) and unnucleated (human) separated erythrocytes.

Determination of vanillylmandelic, 5-hydroxyindoleacetic and homovanillic acid in urine by isocratic liquid chromatography.

A new isocratic HPLC method, employing electrochemical detection, is described for the determination of urinary vanillylmandelic acid, 5-hydroxyindoleacetic acid and homovanillic acid. The main advantages of this technique are: simplicity, simultaneous determination of all analytes, the absence of an extraction procedure, isocratic elution and low cost. The diluted urine is injected onto a C18 reversed phase column. The mobile phase is potassium dihydrogenphosphate buffer containing 1-heptanesulphonic acid, methanol and acetonitrile. The calibration curves are linear from 0.1 to 50 mg/l; the precision data show CV less than 2.36% for within-day assay and less than 2.72% for day-to-day assays. The mean recoveries for supplemented samples are 98.2 to 102.0% for vanillylmandelic acid, 99.6 to 103.9% for 5-hydroxyindoleacetic acid and 98.7 to 102.0% for homovanillic acid. In comparisons of the present method with Radjaipur's extraction method (Radjaipur M. et al., Eur J Clin Chem Clin Biochem 1994; 32:609-13) the slopes for the three analytes were nearly 1, and the confidence region of the intercepts was close to 0. In conclusion the technique seems to be suitable for routine determination of the three analytes, especially for mass screening purposes.

Age-dependent changes of serum oxygen radical scavenger capacity and haemoglobin glycosylation in non-insulin-dependent diabetic patients.

BACKGROUND: Contradictory results have been reported in the literature concerning the correlation between glycosylated haemoglobin (HbA1c) and peroxidation level in serum of diabetic patients. OBJECTIVE: To evaluate this correlation in type 2 diabetic patients by comparing the level of HbA1c with the oxygen radical absorbance capacity (ORAC(OH)) of serum. METHODS: One hundred and five type 2 diabetic patients were enrolled for the study. After having obtained informed consent, venous blood samples were drawn after overnight fast at the time of routine diabetic check-ups. The blood was collected in plain and EDTA (1 mg/ml) tubes. Glycosylated haemoglobin (HbA1c) was determined by cation-exchange chromatography (HPLC), and spectrophotometric detection (Diamat Analyzer, BioRad). Serum was used for biochemical determinations performed by standard laboratory procedures and for ORAC(OH) analysis. This last parameter was determined measuring the loss of beta-phycoerytrin fluorescence due to oxidation by hydroxyl radicals generated by Cu(2+) and H(2)O(2), in the presence and absence of serum. Seventy-eight control age-matched subjects were obtained from the personnel staff of our Research Department and old healthy subjects, selected on the basis of Senieur Protocol, were relatives of the above mentioned personnel. RESULTS: When the population of diabetic patients was taken as a whole, a decrease of ORAC(OH) has been observed compared to the controls. Moreover, negative correlations were found comparing ORAC(OH) either with HbA1c (r = -0.213; p = 0.029) and with the age of patients (r = -0.27; p = 0.005). To better understand the effect of age, the data were re-examined dividing the diabetics into two populations, i.e. under and over 65 years of age. An age-dependent decrease of ORAC(OH) and an increase in HbA1c levels has been observed comparing these two populations; however, the correlation between the two parameters remained statistically significant only in the oldest group (r = -0.31; p = 0.026). CONCLUSIONS: Present data point to an involvement of oxidative stress in the glycation of haemoglobin especially in old diabetic patients, and provide support for the potential use of an antioxidant therapy in these patients, irrespective of their glycaemic control.