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.

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.

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.

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.

Relationship between blood metals and inflammation in taxi drivers.

BACKGROUND: Cardiovascular disease is a cause of concern in public health worldwide, reinforcing the need for studies related to the identification of potential agents that contribute to the inflammation process and atherosclerosis. This study aimed to evaluate whether metals are associated with inflammatory and kidney damage and could contribute to the atherosclerosis process. METHODS: Blood metals, inflammatory markers, homocysteine, antioxidants and renal markers were measured in 42 taxi drivers and 27 controls (non-occupationally exposed). RESULTS: Taxi drivers had increased Hg, As, Pb and Cd levels, however Cu and Zn levels were decreased compared to controls (p<0.05). Hg, As and Pb levels were positively associated with pro-inflammatory cytokines, nitric oxide and negatively associated with glutathione peroxidase. Moreover, Hg, As and Pb presented positive associations with homocysteine, an independent risk factor for atherosclerosis. Regarding markers of kidney function, N-acetyl-beta-d-glucosaminidase levels were increased in taxi drivers and correlated to inflammation markers. CONCLUSION: Hg levels were found above the recommended limits in taxi drivers and both Hg and As levels showed associations with inflammatory process, oxidative status and homocysteine. Thus, chemical substances as Hg and As can be considered as additional contributors to the development of cardiovascular diseases.

Iron metabolism in hamsters experimentally infected with Leptospira interrogans serovar Pomona: influence on disease pathogenesis.

The aim of this study was to analyze the classic iron markers associated to the storage process in hamsters experimentally infected by Leptospira interrogans serovar Pomona. Four groups with six hamsters each were used; two were negative controls (C7 and C14) and two were composed by infected animals (T7 and T14). Blood samples were collected on the seventh (C7 and T7) and fourteenth days (C14 and T14) post-inoculation. Iron availability was determined in sera samples through the assessment of iron, ferritin, transferrin, and iron binding capacity, whereas the bone marrow was also evaluated for the presence of iron by Pearl's reaction. Additionally, the total antioxidant capacity (TAC) and total oxidant status (TOS) were assessed, along with hepcidin and IL-6 levels. Based on the results, it was possible to observe the onset of an anemic profile, predominantly hemolytic and regenerative. Also, The other parameters showed an increase in seric iron (P<0.01) and ferritin (P<0.01), and a positive Pearl's reaction in T7 and T14, when compared with the control groups. Transferrin levels decreased (P<0.05) in animals of T14 with saturation index. TAC was increased in both periods (P<0.01), while TOS was increased only on T14 (P<0.05). Hepcidin and IL-6 were increased on T7 and T14 (P<0.01). Therefore, it was observed that the serum profile from infected animals showed a strong hemolytic pattern, with some demonstration of ferric tissue sequestration when the infection tended to become chronic. The results show that iron metabolism is activated in hamsters infected by L. interrogans serovar Pomona.

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.

Antinociceptive and antiedematogenic effect of pecan (Carya illinoensis) nut shell extract in mice: a possible beneficial use for a by-product of the nut industry.

Abstract Background: Interest in pecan (Carya illinoensis) nut shells, a by-product of the nut industry, has increased due to its anti-inflammatory and antioxidant activities. The goal of this study was to evaluate the antinociceptive and antiedematogenic activity and the mechanisms of the pecan shell aqueous extract (AE). Methods: First, we performed fingerprinting of C. illinoensis AE. The antinociceptive and antiedematogenic effects of AE intragastric (i.g.) administration in mice (male Swiss mice 20-30 g) were evaluated using the acetic acid test or after subcutaneous (s.c.) paw injection of diverse transient receptor potential ankyrin 1 (TRPA1) agonists, including hydrogen peroxide (H2O2), allyl isothiocyanate, or cinnamaldehyde. We also observed AE antinociceptive and antiedematogenic effects after carrageenan s.c. paw injection and measured H2O2 production. Moreover, we observed the development of adverse effects after AE i.g. treatment. Results: The high-performance liquid chromatography fingerprinting of AE showed the presence of rutin. AE or rutin i.g. treatment produced antinociception in the acetic acid test and reduced the nociception and edema mediated by H2O2 s.c. hind paw injection or nociception induced by other TRPA1 agonists. Moreover, AE or rutin reduced the hyperalgesia, edema, and H2O2 production induced by carrageenan s.c. paw injection. No motor, gastric, or toxicological alterations were observed after AE administration. Conclusions: Collectively, the present results show that AE and its constituent rutin produced antinociceptive and antiedematogenic action in models of acute and persistent inflammatory nociception and it seems to be related to the inhibition of TRPA1 receptor activation.

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.

Assessment of urinary γ-glutamyltransferase and alkaline phosphatase for diagnosis of diabetic nephropathy.

BACKGROUND: Urinary biomarkers of tubular damage can be useful for early diagnosis of diabetic nephropathy. Thus, the aim of this study was to test the diagnostic accuracy of the urinary excretion of γ-glutamyltransferase (GGT) and alkaline phosphatase (ALP) for diagnosis of diabetic nephropathy (DN). METHODS: Fasting glucose, fructosamine, serum creatinine, glomerular filtration rate (GFR), serum uric acid, serum albumin, and urinary albumin, creatinine, GGT and ALP were assessed in 74 type 2 diabetic patients without nephropathy and 38 type 2 diabetic patients with nephropathy. RESULTS: Urinary GGT and ALP were threefold higher in type 2 diabetic patients with nephropathy. Significant correlations were observed between urinary albumin and GGT (r=0.439, P<0.001) and urinary albumin and ALP (r=0.305, P<0.01). Areas under the curve for GGT and ALP were 0.7696 (P<0.001) and 0.7233 (P<0.001), respectively. At a cut-off value of 72U/g creatinine, GGT demonstrated a sensitivity of 96.0% and a specificity of 52.6%. At a cut-off value of 20U/g creatinine, ALP demonstrated a sensitivity and specificity of 83.8% and 36.8%, respectively. CONCLUSIONS: Urinary GGT and ALP have potential value in the diagnosis of nephropathy in type 2 diabetic patients, but GGT has a slightly higher ability to discriminate nephropathy than ALP.