thiobarbituric-acid and Diabetes-Mellitus

Berberis vulgaris is a well known plant with traditional herbal medical history. The aims of this study was to bioscreen and compare the in vitro biological activity (antioxidant, cholinergic, antidaibetic and the anticancer) of barberry crude extract and berberine active compound.. The effect of B. vulgaris extract and berberine chloride on cellular thiobarbituric acid reactive species (TBARS) formation, diphenyle-α-picrylhydrazyl (DPPH) oxidation, cellular nitric oxide (NO) radical scavenging capability, superoxide dismutase (SOD), glutathione peroxidase (GPx), acetylcholinesterase (AChE) and α-gulcosidase activities were spectrophotometrically determined. On the other hand, the effect of extract and berberine as anticancer was estimated on three different cell lines which were MCF-7, HepG-2, and Caco-2 cells by using neutral red uptake assay which compared with control normal cells (PBMC).. Our results showed that barberry crude extract contains 0.6 mg berberine/mg crude extract. Barberry extract showed potent antioxidative capacity through decreasing TBARS, NO and the oxidation of DPPH that associated with GPx and SOD hyperactivation. Inhibitory effect of berberis crude extract on α-glucosidase was more potent than that of berberine chloride, while both had the same AChE inhibitory effect. Besides, different concentrations of both berberine chloride and barberry ethanolic extract showed to have no growth inhibitory effect on normal blood cells (PBMC). Otherwise, both berberine chloride and barberry ethanolic extract showed to have inhibitory effect on the growth of breast, liver and colon cancer cell lines (MCF7, HepG2 and CACO-2, respectively) at different incubation times starting from 24 hrs up to 72 hrs and the inhibitory effect increased with time in a dose dependent manner.. This work demonstrates the potential of the barberry crude extract and its active alkaloid, berberine, on suppressing lipid peroxidation, suggesting a promising use in the treatment of hepatic oxidative stress, Alzheimer and idiopathic male factor infertility. Beside, berberis vulgaris ethanolic extract is safe non-toxic extract as it was not inhibit the growth of PBMC that can induce cancer cell death that could return to its powerful antioxidant activity.

Topics: Acetylcholinesterase; alpha-Glucosidases; Animals; Antineoplastic Agents; Antioxidants; Berberine; Berberis; Cell Line, Tumor; Cell Survival; Cholinesterase Inhibitors; Chromatography, High Pressure Liquid; Diabetes Mellitus; Ethanol; Glutathione Peroxidase; Glycoside Hydrolase Inhibitors; Humans; Leukocytes, Mononuclear; Lipid Peroxidation; Male; Mice; Mice, Inbred BALB C; Oxidation-Reduction; Oxidative Stress; Plant Extracts; Plant Roots; Superoxide Dismutase; Thiobarbiturates

Diabetes is associated with a high incidence of erectile dysfunction (ED) and poor response to standard treatments. Oxidative stress could be relevant in the pathophysiology of diabetic ED.. To evaluate the effects of the antioxidant, AC3056 (2,6-di-t-butyl-4-((dimethyl-4-methoxyphenylsilyl)methyloxy)phenol), on diabetic ED.. Erectile responses to cavernosal nerve electrical stimulation were determined in streptozotocin-induced diabetic rats. Relaxation of human corpus cavernosal (HCC) tissue and penile resistance arteries (HPRA) from human cavernosal specimens was evaluated in organ chambers and myographs, respectively.. The influence of AC3056 on erectile responses, lipid peroxidation, and nitrite plus nitrate serum content, and nuclear factor-kappaB (NF-kappaB) expression in penile tissue, in diabetic rats, and on endothelium-dependent and neurogenic relaxation of HCC and HPRA from diabetic patients was determined.. Eight weeks of diabetes caused ED in rats that was prevented by oral AC3056 (0.3% w/w in rat chow) when given from the induction of diabetes. AC3056 also prevented the diabetes-induced elevation of serum thiobarbituric acid-reactive substances (TBARS), the reduction of serum nitric oxide (NO) derivatives, and the increase of NF-kappaB expression. Acute oral administration of AC3056 (450 mg/kg) partially reversed ED in 8-week diabetic rats. Complete reversion of ED was achieved after 3 days of treatment with 0.3% AC3056. This effect remained after 5 weeks of treatment, but it disappeared after withdrawing for 1 week. Erectile function in diabetic rats was inversely related to serum TBARS. AC3056- (30 microM) reversed endothelial dysfunction in diabetic HCC and enhanced endothelium-dependent relaxation in diabetic HPRA and significantly potentiated neurogenic relaxation of both tissues. The reduced cGMP content in HCC from diabetic patients after exposure to acetylcholine (10 microM) was corrected by AC3056 (30 microM).. These results suggest that oxidative stress has a relevant role in pathophysiology of diabetic ED and provide a rationale for the use of antioxidant therapy in the treatment of ED in diabetes.

Topics: Animals; Antioxidants; Blotting, Western; Diabetes Mellitus; Disease Models, Animal; Drug Administration Schedule; Erectile Dysfunction; Humans; Lipid Peroxidation; Male; Nitric Oxide; Organosilicon Compounds; Penis; Rats; Rats, Sprague-Dawley; Thiobarbiturates

Glutathione (GSH) plays an important role in the cellular defense against (per-)oxidative stress. The capacity of this cellular defense system may be related to the oxygen tension, cells are normally subjected to in vivo; therefore, we studied the de novo synthesis of glutathione, and the redox turnover under peroxidative stress, in human umbilical vein and artery endothelial cells (HUVEC, HUAEC) and human skin fibroblasts. De novo synthesis in these cell types was studied in vitro by measuring the time course of intracellular GSH recovery after depletion with diamide. For fibroblasts, the initial rate of de novo synthesis after GSH depletion was twice that of the endothelial cell strains. In the endothelial cells (HUVEC, HUAEC) the original intracellular GSH level is reached within 40 min. while in the same time span, the GSH level in fibroblasts returned to 75% of control level. The activity of the hexose monophosphate shunt (HMS) was determined under oxidative stress as a measure for the coupled redox turnover of intracellular GSH. Under control conditions the HMS in endothelial cells was twice as high as in fibroblasts. Cumene hydroperoxide (40 microM) induced a three-fold increase in HMS in both HUVEC and HUAEC, while fibroblasts exhibited an increase of 83%. During the same peroxidative stress, the intracellular GSH concentration of HUVEC, HUAEC and fibroblasts stayed at control level. So with respect to GSH metabolism there were no differences between the two endothelial cell strains. In comparison with the endothelial cells, the fibroblasts were less susceptible toward oxidative stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Benzene Derivatives; Cells, Cultured; Diabetes Mellitus; Endothelium, Vascular; Free Radicals; Glutathione; Humans; Hyperlipoproteinemias; Oxidation-Reduction; Pentose Phosphate Pathway; Stress, Physiological; Thiobarbiturates

Patients with transient ischemic attacks (TIA) were previously shown to have high plasma values of thiobarbituric acid-reactive substances (TBA-RS). To study whether these changes could be related to platelet activability, TBA-RS was investigated in 24 TIA patients before and 24 h after 1 g aspirin, an inhibitor of platelet cyclooxygenase pathway. Baseline TBA-RS values were significantly higher in TIA than in controls. Conversely, TIA patients had TBA-RS values after aspirin similar to controls, suggesting that the increase of plasma TBA-RS was not attributable to platelet hyperfunction. The evaluation of metabolic profile showed that patients with highest TBA-RS had hypercholesterolemia, hypertriglyceridemia, and/or diabetes mellitus. This study suggests that the increase of plasma TBA-RS in TIA could be an epiphenomenon of altered metabolic pathway.

Topics: Administration, Oral; Aged; Arteriosclerosis; Aspirin; Blood Glucose; Blood Platelets; Coronary Disease; Diabetes Mellitus; Female; Humans; Hypercholesterolemia; Ischemic Attack, Transient; Lipid Peroxides; Male; Middle Aged; Thiobarbiturates

We glycated immunoglobulins from commercial kits designed to measure human ferritin, thyrotropin, and transferrin, and compared the calibration curves for assays utilizing glycated antibodies with those of assays utilizing non-glycated antibodies. Glycation was verified by borate affinity chromatography and assay with thiobarbituric acid reagent. We found no evidence that antigen-antibody binding is impaired by nonenzymatic glycation of antibodies. Our results provide no evidence in support of the supposition that glycation may be a contributory factor in the decreased resistance of diabetics to infection.

Topics: Antibodies; Antigen-Antibody Reactions; Antigens; Chromatography, Affinity; Diabetes Mellitus; Ferritins; Glucose; Glycosylation; Humans; Immunoglobulins; Reagent Kits, Diagnostic; Thiobarbiturates; Thyrotropin; Transferrin

Topics: Blood Proteins; Colorimetry; Diabetes Mellitus; Fructose; Glycated Serum Proteins; Glycoproteins; Humans; Nitroblue Tetrazolium; Thiobarbiturates

Topics: Colorimetry; Diabetes Mellitus; Evaluation Studies as Topic; Glycated Hemoglobin; Humans; Hydrolysis; Thiobarbiturates

Techniques for affinity measurement of glycated albumin and for glycated total plasma protein have been developed. The two techniques were contrasted. Both techniques are linear over a 100-fold range of sample concentrations. There appears to be a non-specific early glucose binding phase to non-albumin plasma proteins. Although this phase is detected by radioactive incorporation and thiobarbituric acid, it does not interfere with the affinity determination, which does not appear to detect the early binding species. The correlation of glycated albumin levels with glycated hemoglobin levels is much stronger than that of glycated globulin levels with glycated hemoglobin levels. Due to the large contribution of glycated albumin levels to total glycated serum protein levels, the correlation of the latter with glycated hemoglobin levels is sufficiently strong to allow the use of either technique as an adequate index of glycation.

Topics: Boronic Acids; Chromatography, Affinity; Diabetes Mellitus; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Protein Binding; Serum Albumin; Thiobarbiturates; Tritium

Two techniques originally developed for measurement of glycated ("glycosylated") hemoglobin but also applicable to determination of glycated albumin are the thiobarbituric acid colorimetric technique (I) and the aminophenylboronic acid affinity chromatographic procedure (II). The latter reliably distinguishes diabetics from nondiabetics, and concentrations of glycated hemoglobin and glycated albumin are linearly correlated. I is nonspecific; it neither correlates with diabetic status nor with values derived via the affinity technique. Most of the chromogenic material is present in the fraction of albumin that does not bind to aminophenylboronic acid. Glucose interferes significantly with I but only slightly with II. Prolonged incubation of plasma with glucose dramatically increases the II-determined glycated albumin. Reactivity with thiobarbituric acid increases much less, and mainly in the II-bound fraction. This fraction contains a high proportion of nonspecifically reactive material. The percentage of glycated albumin determined in crude plasma samples by II differs only slightly from the value determined by purifying the albumin from the plasma. This technique appears more promising than I for eventual clinical applications.

Topics: Blood Glucose; Boronic Acids; Chromatography, Affinity; Colorimetry; Diabetes Mellitus; Electrophoresis; Glycated Hemoglobin; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Serum Albumin; Thiobarbiturates

Assessment of diabetic glycaemic control by nonenzymatically glycosylated albumin (G-A) was investigated. Serum albumin was purified by affinity chromatography on Affi-Gel Blue. Using the purified serum albumin in normal and diabetic subjects, G-A was estimated by a colorimetric thiobarbituric acid (TBA) method and the results compared with the levels determined using aminophenyl boronic acid (PBA) affinity chromatography. There was an excellent correlation between the levels estimated by TBA method and PBA affinity chromatography method (r = 0.94). The levels of G-A increased in patients with poorly controlled diabetes mellitus compared to normals. There was a significant correlation (r = 0.84) between the G-A and HbA1 levels in 43 normal and 167 diabetic subjects. As an estimate of diabetic glycaemic control by G-A, the correlations between the G-A and mean fasting blood sugar (FBS) levels were studied. There was a higher correlation (r = 0.67) between G-A and the mean FBS within 2 weeks. On starting insulin therapy in 8 juvenile diabetic subjects, there was a different temporal relationship between the FBS, G-A and HbA1 levels. The G-A levels were significantly decreased at 1, 2, 3 and 4 weeks compared to the HbA1 levels. The present results indicate that the G-A may provide a valuable tool for assessing the mean blood sugar levels between shorter intervals, since the turnover of serum albumin is considerably faster than that of HbA1.

Topics: Blood Glucose; Boronic Acids; Chromatography, Affinity; Colorimetry; Diabetes Mellitus; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Serum Albumin; Thiobarbiturates

Four assays, high pressure liquid chromatography, colorimetric with thiobarbituric acid, affinity columns, and microcolumn cation exchange were compared for ability to discriminate between samples taken from diabetic and normal subjects; correlation with each other; stability over time at different temperatures; and reproducibility between laboratories. The most discriminatory (10 samples from a diabetic and 10 samples from a normal group) was the microcolumn cation exchange method (t = 5.25; p less than 0.001), but all were significantly different at p less than 0.005. The intra-assay coefficient of variation was 1%-6%, except for the affinity column method which was 13% in normal subjects. High pressure liquid chromatography was used as a reference and the other assays correlated well (r = 0.93-0.99). Storage at -80 degrees C, -20 degrees C, 4 degrees C, and 24 degrees C showed marked differences. The thiobarbituric acid method results were stable except for 24 degrees C. Microcolumn cation exchange was labile under all conditions. Affinity column was stable for up to 15 days, only if samples were stored as whole blood. High pressure liquid chromatography showed an increase in haemoglobin A1a + b and a decrease in the haemoglobin A1c. Haemoglobin A1c was reproducible for 4 days when stored at 4 degrees C and up to 11 days when stored at -80 degrees C. Samples exchanged between centres at 4 degrees C and performed within 5 days by high pressure liquid chromatography for haemoglobin A1 and haemoglobin A1c correlated well (r = 0.98 and 0.99). Samples exchanged between centres after storage (up to 40 days -80 degrees C) correlated (r = 0.99) by the thiobarbituric acid method.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Chromatography, Affinity; Chromatography, High Pressure Liquid; Colorimetry; Diabetes Mellitus; Evaluation Studies as Topic; Glycated Hemoglobin; Humans; Reference Standards; Thiobarbiturates

Boronate affinity chromatography and ion exchange chromatography were used to measure the levels of glycosylated hemoglobins in normal and diabetic hemolysates, as well as the distribution of glucose adducts on alpha-NH2-valine and epsilon-NH2-lysine residues. When analyzed by ion exchange chromatography on BioRex 70 resin, the Hb Alc peak comprised 4.4 +/- 0.6% of 15 normal hemolysates and 9.1 +/- 2.1% of 15 diabetic hemolysates. The "Hb Alc" was rechromatographed on GlycoGel B boronate affinity resin that binds vicinal hydroxyl groups of covalently linked sugars. Only 70 +/- 5% of the hemoglobin adhered to the resin. Analysis by the thiobarbituric acid colorimetric test confirmed that the affinity resin effectively separated glycosylated from nonglycosylated hemoglobin. When corrected for nonglycosylated contaminants, the mean level of Hb Alc in normal hemolysates was 2.9 +/- 0.4%, a value considerably lower than those previously reported. In addition to Hb Alc, 5.2 +/- 0.5% of the remaining hemoglobin (Hb Ao) was glycosylated. In diabetics, glycosylated Ao was increased in parallel with Hb Alc. After reduction with [3H]borohydride and acid hydrolysis, glycosylated amino acids were first purified on Affi-Gel boronate affinity resin and then analyzed by ion exchange chromatography. The glucose adducts on Hb Ao were distributed as follows: alpha-chain N-terminal valine, 14%; alpha-chain lysines, 40%; beta-chain lysines, 46%. This study has revealed several pitfalls in the analysis of nonenzymatically glycosylated proteins. Peaks isolated by ion exchange chromatography or electrophoresis are likely to be contaminated by nonglycosylated proteins. Furthermore, both the thiobarbituric acid test and [3H]borohydride reduction show variable reactivity depending upon the site of the ketoamine-linked glucose.

Topics: Blood Proteins; Borohydrides; Chromatography, Affinity; Chromatography, Ion Exchange; Colorimetry; Diabetes Mellitus; False Positive Reactions; Glycated Hemoglobin; Humans; Lysine; Oxidation-Reduction; Thiobarbiturates; Valine