Etiane Tatsch

A simple and inexpensive automated technique for measurement of serum nitrite/nitrate

OBJECTIVE We described an automated technique for measurement of serum nitrite/nitrate (NO(x)) using the Cobas Mira clinical chemistry analyzer. DESIGN AND METHODS NO(x) was measured by the modified Griess method. Precision, accuracy, linearity, instrument carry-over and lower limit of quantitation (LLOQ) were assessed. RESULTS The automated technique for measurement of serum NO(x) was linear, precise, and accurate. It has a LLOQ of 2.0 μmol/L. CONCLUSION Serum NO(x) measured by the modified Griess method can be applied easily to the Cobas Mira clinical chemistry analyzer.

Diabetic nephropathy: Traditional to proteomic markers

Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes and it is defined as a rise in the urinary albumin excretion (UAE) rate and abnormal renal function. Currently, changes in albuminuria are considered a hallmark of onset or progression of DN. However, some patients with diabetes have advanced renal pathological changes and progressive kidney function decline even if urinary albumin levels are in the normal range, indicating that albuminuria is not the perfect marker for the early detection of DN. The present article provides an overview of the literature reporting some relevant biomarkers that have been found to be associated with DN and that potentially may be used to predict the onset and/or monitor the progression of nephropathy. In particular, biomarkers of renal damage, inflammation, and oxidative stress may be useful tools for detection at an early stage or prediction of DN. Proteomic-based biomarker discovery represents a novel strategy to improve diagnosis, prognosis and treatment of DN; however, proteomics-based approaches are not yet available in most of the clinical chemistry laboratories. The use of a panel with a combination of biomarkers instead of urinary albumin alone seems to be an interesting approach for early detection of DN, including markers of glomerular damage (e.g., albumin), tubular damage (e.g., NAG and KIM-1), inflammation (e.g., TNF-α) and oxidative stress (e.g., 8-OHdG) because these mechanisms contribute to the development and outcomes of this disease.

Assessment of Inflammatory and Oxidative Biomarkers in Obesity and Their Associations with Body Mass Index

The aim of this study was to evaluate the inflammatory and oxidative biomarkers’ levels in obese subjects and their associations with body mass index (BMI), in order to investigate the role of these biomarkers in obesity. Fasting glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, apolipoprotein A, apolipoprotein B, albumin, urinary albumin, creatinine, glomerular filtration rate, interleukin-6 (IL-6), nitrate/nitrite (NOx), and ischemia-modified albumin (IMA) were measured in 93 subjects divided according to different BMI. IL-6, urinary albumin, and IMA levels were significantly higher in obese subjects. However, the levels of NOx were significantly lower in this population. Significant correlations between BMI and IL-6 (r = 0.326, P = 0.002), NOx (r = −0.249, P = 0.021), urinary albumin (r = 0.270, P = 0.008), and IMA (r = 0.286, P = 0.005) were reported. We have shown an increase of IL-6, urinary albumin, and IMA combined with lower levels of NOx in obese patients and an association between of these biomarkers with BMI, suggesting a possible interplay of oxidative stress, inflammation, and endothelial dysfunction state in obesity.

Association between DNA strand breakage and oxidative, inflammatory and endothelial biomarkers in type 2 diabetes

Evidence has been presented recently that type 2 diabetes patients have an increased level of DNA damage. This DNA damage could be associated with oxidative, inflammatory, and endothelial biomarkers and could represent a possible indication of injury in the endothelium and induction of inflammation in type 2 diabetes. To confirm this possible association, DNA strand breakage was evaluated by use of the comet assay and its association with oxidative, inflammatory, and endothelial biomarkers in type 2 diabetes patients. A case-control study (30 healthy controls and 32 subjects with type 2 diabetes) was performed to evaluate the association between DNA damage and NOx (nitrate/nitrite), interleukin-6 (IL-6), urinary albumin, fasting glucose, and glycated hemoglobin (HbA(1c)) levels. Type 2 diabetes patients presented higher DNA damage than control subjects, higher levels of IL-6 and urinary albumin, and lower NOx. Significant correlations between DNA damage and NOx (r=-0.303, p=0.016), IL-6 (r=0.845, p<0.001), urinary albumin (r=0.496, p<0.001), fasting glucose (r=0.449, p<0.001), and HbA(1c) (r=0.575, p<0.001) were reported. Our findings showed an increase of DNA damage in type 2 diabetes especially in those patients with poor glycemic control and associations among NOx, IL-6 and urinary albumin levels with DNA damage.

Urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin as indicators of tubular damage in normoalbuminuric patients with type 2 diabetes.

OBJECTIVES Renal dysfunction has been reported in normoalbuminuric patients, demonstrating the necessity to improve the diagnostic and prognostic tools for diabetic kidney disease (DKD) investigation. Therefore, the aim of this study was to investigate whether the urinary levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are increased in type 2 diabetes mellitus (DM) patients with normal or mildly increased albuminuria. DESIGN AND METHODS In this study, 117 type 2 DM patients classified into three groups according to urinary albumin/creatinine ratio (uACR): uACR<10mg/g creatinine, uACR 10-30mg/g creatinine and uACR>30mg/g creatinine were enrolled. Urinary concentrations of KIM-1 (uKIM-1) and NGAL (uNGAL) were measured. RESULTS uKIM-1 levels increased progressively from uACR<10mg/g creatinine (69.0±20.8pg/ml) to uACR 10-30mg/g creatinine (106.1±41.2pg/ml) and to uACR>30mg/g creatinine (166.0±31.9pg/ml) (P<0.001). In addition, uNGAL levels increased progressively from uACR<10mg/g creatinine (29.5±8.8ng/ml) to uACR 10-30mg/g creatinine (51.7±10.9ng/ml) and to uACR>30mg/g creatinine (71.0±9.6ng/ml) (P<0.001) patients. Similarly, both uKIM-1 and uNGAL adjusted by urinary creatinine were increased in patients with uACR 10-30mg/g creatinine. Significant and positive correlations were observed between uACR, uKIM-1 and uNGAL. CONCLUSIONS uKIM-1 and uNGAL were increased in type 2 DM patients with normal or mildly increased albuminuria, which indicates that tubular and glomerular injuries may be occurring even at the earliest stage of DKD.

Antinociceptive effect of Mirabilis jalapa on acute and chronic pain models in mice.

ETHNOPHARMACOLOGICAL RELEVANCE The infusion or decoction of Mirabilis jalapa leaves is used in traditional medicine in Brazil to treat inflammatory and painful diseases. Thus, the present study was designed to investigate whether the leaf ethyl acetate (Eta) fraction from Mirabilis jalapa exhibits antinociceptive effect in clinically relevant pain models in mice. Furthermore, we have investigated the role of cholinergic system in the antinociceptive action produced by Eta in mice. MATERIALS AND METHODS The effect of Eta administered orally (10mg/kg, p.o.) in mice was verified on the painful hypersensitivity (mechanical allodynia) in models of chronic inflammation (subcutaneous injection of complete Freunds Adjuvant-CFA in the plantar surface of the right hind paw), postoperative (paw surgical incision) and neuropathic (partial sciatic nerve ligation) pain. In the chronic inflammation model, we further verified the effect of Eta treatment on paw edema and interleukin-1β (IL-1β) levels. We also investigated the role of muscarinic and nicotinic receptors in the antiallodynic action produced by Eta as well as the possible action of Eta on in vitro and ex vivo acetylcholinesterase activity in CFA treated animals. Furthermore, we verified the effect of Eta treatment on the parameters of liver and kidney lesion (level of urea, and activity of aspartate aminotransferase and alanine aminotransferase). RESULTS Eta produced marked reduction in the allodynia caused by CFA, surgical incision and partial sciatic nerve ligation. However, Eta did not alter the paw edema or the increase of IL-1β levels produced by CFA. The antinociceptive effect of Eta was reversed by the pre-treatment of animals with the antagonists of muscarinic (atropine, 5mg/kg, s.c) or nicotinic (mecamylamine, 0.001mg/kg, s.c.) receptors. Eta did not alter in vitro acetylcholinesterase activity in blood or spinal cord samples, but it reversed the increase in the acetylcholinesterase activity observed in the spinal cord samples from mice injected with CFA. Moreover, Eta did not alter the indicators of liver or kidney lesion. CONCLUSIONS Based on its use in traditional medicine, the results of the present study confirmed the antinociceptive properties of Eta in clinically relevant pain models. Also its effect on the CFA-induced chronic inflammation seems to be related to acetylcholinesterase inhibition and cholinergic system.

Oxidative DNA damage is associated with inflammatory response, insulin resistance and microvascular complications in type 2 diabetes

Urinary markers of nucleic acid oxidation may be useful biomarkers in diabetes. It has been demonstrated that T2DM patients have an increased level of oxidative DNA damage; however, it is unclear whether increased DNA damage may be related to a greater degree of inflammation and insulin resistance. Thus, the aim of this present study was to investigate the relation of the impact of oxidative DNA damage, assessed by urinary 8-OHdG, on the levels of inflammatory cytokines, as well as insulin resistance. In addition, we also investigated the diagnostic ability of urinary 8-OHdG in the identification of microvascular complications in T2DM.A case-control study, enrolling 22 healthy controls and 54 subjects with T2DM, was performed to evaluate the relation between oxidative DNA damage and interleukin-6 (IL-6), IL-1,tumor necrosis factor-alpha (TNF-α), IL-10, and Homeostasis Model Assessment (HOMA-IR) index. T2DM patients presented higher urinary 8-OHdG, IL-6, IL-1, TNF-α levels and HOMA-IR, and lower IL-10 levels than control subjects. Moreover, urinary 8-OHdG levels were significantly higher in the group T2DM with microvascular complications when compared to the without complications. The areas under the curve for urinary 8-OHdG and urinary albumin were, respectively, 0.836 (P<0.001) and 0.786 (P=0.002). Thus, urinary 8-OHdG has a slightly higher ability to discriminate microvascular complications in T2DM compared with urinary albumin. It was also demonstrated that T2DM patients with higher median of urinary 8-OHdG had significantly elevated levels of IL-6, TNF-α and HOMA-IR, and decreased IL-10 levels. Our findings showed that T2DM patients with higher urinary 8-OHdG levels showed a greater inflammatory degree and higher insulin resistance. It is possible to speculate that T2DM patients present a cascade of events as increasing metabolic abnormalities such as insulin resistance and inflammatory activation, as well as increased ROS generation factors that may contribute directly to greater oxidative DNA damage.

Assessment of Oxidative, Inflammatory, and Fibrinolytic Biomarkers and DNA Strand Breakage in Hypercholesterolemia

The aim of this study was to assess the levels of oxidative, inflammatory, and fibrinolytic biomarkers as well as DNA strand breakage in hypercholesterolemic subjects. Fasting glucose, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, uric acid, total protein, albumin, apolipoprotein (Apo) A, Apo B, advanced oxidation protein products (AOPP), increased ischemia-modified albumin (IMA), ―SH, NOx, IL-6, and D-dimer levels were assessed, and DNA strand breakage was evaluated using comet assay in 38 patients with hypercholesterolemia and 20 healthy controls. Total cholesterol, triglycerides, LDL cholesterol, Apo A, Apo B, AOPP, IMA, IL-6, and D-dimer concentrations were significantly higher in subjects with hypercholesterolemia. However, NOx and plasma ―SH group concentrations were lower in hypercholesterolemic subjects, while no significant differences were observed with respect to DNA strand breakage between the two groups. Hypercholesterolemia is related to oxidative stress and inflammation. Accordingly, AOPP concentration was higher in subjects with hypercholesterolemia, and we speculate that AOPP can reflect the enhancement of protein oxidation and inflammation.

Butyrylcholinesterase and γ-Glutamyltransferase Activities and Oxidative Stress Markers Are Altered in Metabolic Syndrome, But Are Not Affected by Body Mass Index

Metabolic syndrome (MetS) leads to changes in enzymatic activities, oxidative and inflammatory parameters. Adenosine deaminase (ADA), dipeptidyl peptidase IV (DPP-IV), butyrylcholinesterase (BuChE) and γ-glutamyltransferase (γ-GT) activities, C-reactive protein (hsCRP) and nitric oxide levels (NOx), as well as oxidative stress markers were analyzed in 39 subjects with MetS and 48 controls. Also, the influence of body mass index (BMI) and anthropometric measurements were evaluated. Disturbances in antioxidant defenses and higher γ-GT and BuChE activities, NOx and hsCRP levels were observed in subjects with MetS. These findings remained associated with MetS after adjustment for BMI, except for hsCRP. ADA was correlated with age, insulin levels and HOMA-IR index in MetS. DPP-IV and total cholesterol (TC), BuChE activity and TC, and VIT C and hsCRP levels also were correlated. The analyzed parameters may reflect the inflammatory state of the MetS, and could contribute to prevention and control of various aspects of this syndrome.

Genoprotective and hepatoprotective effects of Guarana (Paullinia cupana Mart. var. sorbilis) on CCl4-induced liver damage in rats

Abstract Context: Several biological effects of Paullinia cupana (guarana) have been demonstrated, but little information is available on its effects on the liver. Objective: The current study was designed to evaluate the hepatoprotective and genoprotective effects of powder seeds from guarana on CCl4-induced liver injury in rats. Materials and methods: Male Wistar rats were pretreated with guarana powder (100, 300 and 600 mg/kg) or silymarin 100 mg/kg daily for 14 days before treatment with a single dose of CCl4 (50% CCl4, 1 mL/kg, intraperitoneally). Results: The treatment with CCl4 significantly increased the serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In addition, CCl4 increased the DNA damage index in hepatocytes. Guarana in all concentrations was effective in decreasing the ALT and AST activities when compared with the CCl4-treated group. The treatment with guarana decreased DNA damage index when compared with the CCl4-treated group. In addition, the DNA damage index showed a significant positive correlation with AST and ALT. Discussion and conclusion: These results indicate that the guarana has hepatoprotective activity and prevents the DNA strand breakage in the CCl4-induced liver damage in rats.