Low frataxin mRNA expression is associated with inflammation and oxidative stress in patients with type 2 diabetes.

BACKGROUND: The mitochondrial protein frataxin is involved in iron metabolism, as well as regulation of oxidative stress. To elucidate the association of frataxin with the pathophysiology of diabetes, we evaluated the mRNA levels of frataxin in leukocytes of patients with type 2 diabetes (T2D). In addition, we investigated the relation between frataxin mRNA levels, inflammatory cytokines, and oxidative stress biomarkers. METHODS: A study including 150 subjects (115 patients with T2D and 35 healthy subjects) was performed to evaluate the frataxin mRNA levels in leukocytes. We assessed the relation between frataxin and interleukin (IL)-6, IL-1, tumour necrosis factor-alpha (TNF-α), total oxidation status (TOS), total antioxidant capacity (TAC), and serum iron. RESULTS: The frataxin mRNA levels in the T2D group were significantly lower than those in healthy subjects. It was also demonstrated that T2D patients with frataxin mRNA levels in the lowest quartile had significantly elevated levels of serum iron, TOS, and inflammatory cytokines, such as TNF-α, IL-1, and IL-6, while TAC levels were significantly lower in this quartile when compared with the upper quartile. CONCLUSIONS: Our findings showed that T2D patients with low frataxin mRNA levels showed a high degree of inflammation and oxidative stress. It is speculated that frataxin deficiency in T2D patients can contribute to the imbalance in mitochondrial iron homeostasis leading to the acceleration of oxidative stress and inflammation.

Anti-inflammatory effect of geranium nanoemulsion macrophages induced with soluble protein of Candida albicans.

Pelargonium graveolens is a member of the Geraniaceae family and has been used in folk medicine in many countries because of its anti-inflammatory activity. No studies have yet been reported to evaluate the anti-inflammatory activity of a nanoemulsion containing geranium oil (GO) model in macrophages. In this study the anti-inflammatory effect of Geranium nanoemulsion (NEG) macrophages induced with soluble proteins of Candida albicans was investigated. GO presented citronellol (17.74%) and geraniol (14.43%) as main constituents. The characterization in NEG was demonstrated, showing the particle size of 164 ± 3.5 nm, PDI of 0.12 ± 0.006 and zeta potential -10 mV ± 1.7. The MIC obtained for NEG and GO were 3.64 µg ml-1 and 1.82 µg ml-1, respectively. The viability of the macrophages treated with NEG and GO concentrations (1/2 x, 1x and 2x MIC) was evaluated. There was a significant reduction of viability and the MTT assay was not confirmed after the LDH assay. Anti-inflammatory activity was evaluated by determining nitric oxide (NO), cytokines (interleukin IL-1, IL-6 and IL-10), tumor necrosis factor-α (TNF) and the expression levels gene of interleukin (IL-2), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The apoptosis inhibition capacity was assessed by determination of INFγ, caspase 3 and caspase 8. The results indicated that there was a significant increase of NO in the levels after treatment with NEG and significantly reduced levels after treatment with GO. The cytokines (IL-1, IL-6, IL-10, and TNF) were evaluated and NEG (½ x, 1x MIC) decreased IL-1 levels by 1.25-1.37 times, respectively. The NEG did not decrease IL-6 levels and a significant increase was observed for IL-10. GO significantly decreased IL-6 and IL-10 levels. There was a significant decrease in IL-2 and COX-2 levels and increased levels of iNOs. The levels of IFNγ and caspase-3 after treatment with NEG decreased indicating an anti-inflammatory effect and can inhibit apoptosis. Finally, the levels of caspase-8 do not change. Thus, pretreatment with NEG induced an anti-inflammatory effect against soluble proteins of C. albicans model macrophages.

Iron metabolism and oxidative profile of dogs naturally infected by Ehrlichia canis: Acute and subclinical disease.

The aim of this study was to evaluate the oxidant profile and iron metabolism in serum of dogs infected by Ehrlichia canis. Banked sera samples of dogs were divided into two groups: negative control (n = 17) and infected by E. canis on acute (n = 24), and subclinical (n = 18) phases of the disease. The eritrogram, leucogram, and platelet counts were evaluate as well as iron, ferritin, and transferrin levels, latent iron binding capacity (LIBC), and transferrin saturation index (TSI) concentration. In addition, the advanced oxidation protein products (AOPP) and ferric reducing ability of plasma (FRAP) in sera were also analyzed. Blood samples were examined for the presence of E. canis by PCR techniques. History and clinical signals were recorded for each dog. During the acute phase of the disease, infected animals showed thrombocytopenia and anemia when compared to healthy animals (P < 0.05) as a consequence of lower iron levels. Ferritin and transferrin levels were higher in both phases (acute and subclinical) of the disease. The AOPP and FRAP levels increased in infected animals on the acute phase; however, the opposite occurred in the subclinical phase. We concluded that dogs naturally infected by E. canis showed changes in the iron metabolism and developed an oxidant status in consequence of disease pathophysiology.

Evaluation of the In Vitro Interference of Hemoglobin, Lipids, and Bilirubin on the Measurement of Plasma Advanced Oxidation Protein Products.

BACKGROUND: The pre-analytical phase is the most vulnerable and contributes to the majority of errors in laboratory assays. Therefore, the aim of this study was to evaluate in vitro interference of hemoglobin, lipids, and bilirubin on measurement of plasma advanced oxidation protein products (AOPPs). METHODS: AOPPs were measured in a sample spiked with increasing concentrations of hemoglobin, Intralipid® 20% or bilirubin. Then, the relative deviation of the result from the nonspiked baseline value was calculated. RESULTS: We found that all interferents analyzed influenced AOPPs baseline value significantly. Hemoglobin produced a non-linear negative bias, with a maximum decrease of 21.1%. Otherwise, lipids and bilirubin produced a positive bias, ranging from 20.0 to 90.9%, and 12.1 to 33.2%, respectively. CONCLUSIONS: The addition of interferents produced a significant bias in the measurement of AOPPs and in different degrees at the majority of concentrations added.

Relationship between DNA damage in liver, heart, spleen and total blood cells and disease pathogenesis of infected rats by Trypanosoma evansi.

Trypanosoma evansi is an important pathogen that causes changes in nitric oxide (NO) levels and antioxidant enzymes, as well as oxidative stress. The present study evaluated the in vivo effect of T. evansi infection on frequency and index of DNA damage in liver, heart, spleen and total blood of rats. Twenty rats were assigned into two groups with ten rats each, being subdivided into four subgroups (A1 and A2, 5 animals/group; and B1 and B2, 5 animals/group). Rats in the subgroups A1 and A2 were used as control (uninfected) and animals in the subgroups B1 and B2 were inoculated with T. evansi (infected). NO in serum and the comet assay were used to measure DNA damage index (DI) and damage frequency (DF) in liver, heart, spleen and total blood of infected rats. Increased NO levels on days 3 and 9 post-infection (PI) was observed (P < 0.001). Also, it was verified an increase on DI and DF in the evaluated organs on days 3 and 9 PI (P < 0.001). Our data show that T. evansi infection causes genotoxicity due to the production of NO, causing not only the death of the protozoan, but also inducing DNA damage in the host.

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

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.

Circulating Double-Stranded DNA in Plasma of Hemodialysis Patients and its Association with Iron Stores.

BACKGROUND: Chronic kidney disease (CKD) is characterized by oxidative stress, and most of the adverse effects of CKD are mediated by iron-catalyzed ROS generation. The DNA, in particular, is more susceptible to attack by ROS than other proteins and membrane lipids. Considering the evidence on the relationship between CKD, iron metabolism, and DNA damage, the purpose of this study was to evaluate cell-free DNA in the plasma of HD patients and its association with iron status biomarkers and kidney function. METHODS: Measurements of the circulating cell-free DNA in plasma, iron, ferritin, transferrin and other biochemical parameters were performed in 40 chronic hemodialysis (HD) patients and 40 healthy controls. Blood samples were also collected 1 hour before and 1 hour after the HD session to check whether a single HD session would be able to promote an increase in cell-free DNA in the plasma. RESULTS: Cell-free DNA in plasma was significantly increased in HD patients in comparison with healthy controls (p = 0.0017), and significant correlations were observed between cell-free DNA and GFR and ferritin. Our findings showed that a single HD session was not able to promote an increase in cell-free DNA. It was reported that increased ferritin levels and reduced GFR were associated with higher circulating cell-free DNA. CONCLUSIONS: The HD patients presented increased ceIl-free DNA. In addition, the increase of ferritin levels and the decrease of GFR were associated with DNA damage. We also observed that a single HD session was not able to promote an increase in cell-free DNA.

Evaluation of the in vitro interference of bilirubin and lipids in the measurement of the plasma glutathione reductase activity.

BACKGROUND: The pre-analytical phase is the most vulnerable to errors, and some of the most common interferents in laboratory routine are bilirubin and lipemia. Therefore, the aim of this study was to evaluate the in vitro interference of bilirubin and lipids in the measurement of the activity of glutathione reductase (GR) in plasma samples. METHODS: The evaluation of the in vitro interference of bilirubin was performed by addition of bilirubin to a plasma pool at the following final concentrations: 0.9, 1.9, 3.8, 7.5, 15, and 30 mg/dL. The turbidity of lipemia was simulated by the addition of Intralipid to the plasma pool at the following final concentrations: 0.67, 1.25, 2.5, 5, and 10 mg/dL. GR activity was measured on a Cobas MIRA automated analyzer. RESULTS: Plasma GR activity was significantly affected by bilirubin and lipids. At the concentrations of 0.9 to 30 mg/dL of bilirubin added, the decrease of GR activity ranged between 22.9 to 45.4%. At the concentrations of 0.67 to 10 mg/dL of Intralipid added, the decrease of GR activity ranged between 22.4 to 36.5%. CONCLUSIONS: The addition of bilirubin and lipids in plasma samples interferes negatively in the measurement of GR activity, since GR values are reduced in the presence of these in vitro interferents.

Assessment of ischemia-modified albumin levels in patients with rheumatoid arthritis.

BACKGROUND: Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Abundant amounts of ROS have been identified in the synovial fluid of RA patients. The accumulation of ROS in cells also serves as an important intracellular signaling of molecules that amplify the synovial inflammatory-proliferative response. Thus, the aim of this study was to assess the IMA levels and other oxidative stress and inflammatory biomarkers in RA subjects. METHODS: IMA, AOPP, CRP, hemoglobin, Hct, MCV, RF, creatinine, urea levels were assessed in 16 RA subjects and 20 healthy controls. RESULTS: IMA levels were significantly higher in the RA group than in the control group (0.495 +/- 0.01 vs. 0.433 +/- 0.02 ABSU, p = 0.038). No significant differences were observed for the other markers studied. CONCLUSIONS: This study demonstrated that RA is related to oxidative stress and inflammation. We also showed for the first time an increase of IMA levels in RA subjects, suggesting that this pathology promotes an increase in the oxidative stress process.

Effect of temperature on albumin cobalt binding and its influence on ischemia-modified albumin levels in patients with suspected acute coronary syndrome.

BACKGROUND: Ischemia-modified albumin (IMA) has been shown to be a rapidly rising and sensitive biochemical marker especially for the diagnosis of myocardial ischemia. The aim of this study was to evaluate the influence of temperature on the capacity of cobalt binding to human albumin and the influence of this variable on IMA measurement. METHODS: The following temperatures of incubation were tested for human albumin standard 25 degrees C, 28 degrees C, 31 degrees C, 34 degrees C, and 37 degrees C and for patients with suspected acute coronary syndrome, room temperature and 37 degrees C. IMA was measured by cobalt-albumin binding assay. RESULTS: There was a strong positive correlation (r = 0.98, p < 0.001) between IMA and the assay temperatures. IMA levels were 0.68 +/- 0.25 absorbance units (ABSU) at room temperature and 0.92 +/- 0.33 ABSU (p < 0.001) at 37 degrees C in the study participants. CONCLUSIONS: IMA values were influenced by the assay temperature.

Hba(1c) as a tool for the diagnosis of type 2 diabetes: comparison with fasting glucose.

BACKGROUND: Glycated hemoglobin (HbA(1c)) has been proposed for the diagnosis of diabetes. However, several countries have not incorporated its use for this purpose yet and there is no consensus on a suitable cut-off point of HbA(1c) for the diagnosis of diabetes. Thus, the aim of this study was to evaluate the diagnostic characteristics of HbA(1c) and fasting plasma glucose (FPG) for the assessment of type 2 diabetes. METHODS: FPG, HbA(1c), and creatinine levels were assessed in 47 patients with type 2 diabetes and 46 healthy controls. RESULTS: The areas under the curve for HbA(1c) > or = 6.5% and FPG > or = 7.0 mmol/L were 0.97 and 0.92, respectively. HbA(1c). has a slightly higher ability to discriminate type 2 diabetes compared with FPG. The association between HbA(1c) and type 2 diabetes was independent of gender, age, hypertension, smoking, and body mass index. CONCLUSIONS: HbA(1c) was able to be used for the diagnosis of type 2 diabetes.

Reference limits of the urinary gamma-glutamyltransferase in a healthy population and effects of short-term storage on the enzyme activity.

BACKGROUND: Gamma-glutamyltransferase (GGT) is present mainly in proximal renal tubule, and urinary GGT is an indicator of tubular damage since it may show renal changes before they are identified by using conventional measurements. Therefore, it is of interest to establish the reference limits of urinary GGT for a healthy population, as well as to investigate the stability of GGT in urine samples stored at 4 °C and -20 °C. METHODS: GGT was assessed in urine samples from 127 healthy patients by use of a reference method based on the 5-Amino-2-Nitrobenzoate formation. Stability of GGT was evaluated in 10 urine samples stored at temperatures of 4 °C and -20 °C for a period up to 4 weeks. RESULTS: Urinary GGT values for healthy volunteers were 14 U/g creatinine for the lower reference limit and 79 U/g creatinine for the upper reference limit. Urinary GGT values were approximately 56% lower in samples stored at -20 °C than fresh samples, while samples stored at 4 °C presented a decrease of 11% in GGT values compared to fresh samples. CONCLUSIONS: Reference limits for urinary GGT in healthy subjects were 14 to 79 U/g creatinine, and it is recommended to measure urinary GGT in fresh specimens.

Inflammation, Oxidation, Caloric Expenditure and Cognitive Impairment in Brazilian Elderly Assisted at Primary Care.

Cognitive impairment (CI) has a multifactorial etiology. Some studies have suggested that inflammatory, oxidative and antioxidant status and physical activity are associated with CI. However, the evidence on this subject is still controversial. The goal of this study was to verify the association of caloric expenditure by physical activity, oxidative, antioxidant power and inflammatory biomarkers with CI in older adults. We performed a cross-sectional study of 424 elderly (224 with normal cognitive function and 200 with CI) patients from the Family Health Strategy in Porto Alegre, Rio Grande do Sul, Brazil. The variables investigated were sociodemographic, biochemical, inflammatory (hs-CRP, IL-6), oxidative (TBARS, AOPP), antioxidant power (FRAP) biomarkers, energy expenditure, and cognitive function. The instruments used were the Minnesota Leisure Time Physical Activity Questionnaire + Compendium of Physical Activities, classification of energy costs of human physical activities (for physical activity evaluation and measurement of energy expenditure in METs), and a battery of neuropsychiatric instruments (for cognitive ability assessment). We found statistically significant differences only with respect to HDL-c and age (higher averages in the CI group; P<0.05). We observed no differences between the groups with respect to biochemical, inflammatory, oxidative and FRAP biomarkers or caloric expenditure. Logistic regression showed that HDL-c (OR=1.02 [IC=95%; 1.01-1.04]; P=0.011), and age (OR=1.05 [IC=95%; 1.02-1.08]; P=0.004) are independent factors associated with CI. Our results suggest that the biochemical (except HDL-c), inflammatory, oxidative, and FRAP biomarkers investigated and caloric expenditure are not associated with CI in the elderly assisted at primary care.

Urinary inflammatory cytokines as indicators of kidney damage in type 2 diabetic patients.

BACKGROUND: The aim of this study was to investigate whether urinary levels of interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) are altered in normoalbuminuric patients with type 2 diabetes mellitus (T2DM), and whether these cytokines are able to identify diabetic kidney disease (DKD) among these patients. METHODS: This study included 125 T2DM patients classified into 3 groups according to urinary albumin/creatinine ratio (uACR): uACR <10mg/g creatinine, uACR 10-30mg/g creatinine and uACR >30mg/g creatinine. Urinary inflammatory cytokines were measured. RESULTS: The urinary IL-6 concentrations increased from uACR <10 (97.2±26.4pg/ml) to uACR 10-30 (113.6±28.0pg/ml) and to uACR >30mg/g creatinine (163.5±25.6pg/ml) (P<0.05 and P<0.001, respectively) patients. The urinary IL-10 concentrations decreased in these uACR ranges [100.0 (58.0-141.0) pg/ml vs. 62.0 (54.5-71.5) pg/ml vs. 42.0 (32.0-48.0) pg/ml] (P<0.05 and P<0.001, respectively). All urinary cytokines demonstrated good ability to identify DKD (areas under curves >0.9). CONCLUSIONS: Urinary inflammatory cytokines, especially IL-6 and IL-10, may assist in the identification of DKD in T2DM patients, even in the absence of micro- and macroalbuminuria.

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.

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.

The association between the chronic use of non-steroidal anti- inflammatory drugs and oxidative and inflammatory markers in the elderly.

OBJECTIVES: Investigate the association between the chronic or occasional use of nonsteroidal anti-inflammatory drugs (NSAIDs) and plasma levels of oxidative and inflammatory markers in elderly at the Family Health Strategy in Brazil. METHODS: It was a cross-sectional study of data collected from random elderly volunteers. A questionnaire including sociodemographic, health and medicine use data was administered. The blood levels of FRAP (ferric reducing ability of plasma), AOPP (plasma advanced oxidation protein products), MDA (malondialdehyde) and insulin were measured. RESULTS: The study sample comprised 758 elderly patients, of which 121 (15.96%) used NSAIDs. The mean age was 68.53 years and 68.41 for individuals who used NSAIDs occasionally and chronically, respectively. Gastric problems may be associated with the chronic or occasional use of NSAIDs (P = 0.042). Which indicates mean plasma levels of Insulin and HOMA-IR (Homeostasis Model Assessment Insulin Resistance) are increased in chronic use of NSAIDs and describes a statistical trend (P = 0.065) for the association of chronic NSAIDs use with the BMI (body mass index) of the subjects studied. CONCLUSION: This study suggests that there is no association between the chronic or occasional use of NSAIDs and oxidative and inflammatory markers. It is known that NSAIDs have innumerable adverse effects, but they can have some benefits. So, additional studies are needed to clarify whether NSAIDs are associated with these markers and whether they are related with their real consequences.