sepharose and Diabetes-Mellitus

Diabetic wounds with complex pathophysiology significantly burden the wound care industry and require novel management strategies. In the present study, we hypothesized that agarose-curdlan based nanofibrous dressings could be an effective biomaterial for addressing diabetic wounds due to their inherent healing properties. Hence, agarose/curdlan/polyvinyl alcohol based nanofibrous mats loaded with ciprofloxacin (0, 1, 3, and 5 wt%) were fabricated using an electrospinning technique with water and formic acid. In vitro evaluation revealed the average diameter of the fabricated nanofibers between 115 and 146 nm with high swelling (~450-500 %) properties. They exhibited enhanced mechanical strength (7.46 ± 0.80 MPa -7.79 ± 0.007 MPa) and significant biocompatibility (~90-98 %) with L929 and NIH 3T3 mouse fibroblasts. In vitro scratch assay showed higher proliferation and migration of fibroblasts (~90-100 % wound closure) compared to electrospun PVA and control. Significant antibacterial activity was observed against Escherichia coli and Staphylococcus aureus. In vitro real-time gene expression studies with human THP-1 cell line revealed a significant downregulation of pro-inflammatory cytokines (8.64 fold decrease for TNF-α) and upregulation of anti-inflammatory cytokines (6.83 fold increase for IL-10) compared to lipopolysaccharide. In brief, the results advocate agarose-curdlan mat as a potential multifunctional, bioactive, and eco-friendly dressing for healing diabetic wounds.

Topics: Animals; Anti-Bacterial Agents; Diabetes Mellitus; Humans; Lipopolysaccharides; Mice; Nanofibers; Polyvinyl Alcohol; Sepharose

Current management for diabetes has stimulated the development of versatile 3D-based hydrogels as in vitro platforms for insulin release and as support for the encapsulation of pancreatic cells and islets of Langerhans. This work aimed to create agarose/fucoidan hydrogels to encapsulate pancreatic cells as a potential biomaterial for diabetes therapeutics. The hydrogels were produced by combining fucoidan (Fu) and agarose (Aga), marine polysaccharides derived from the cell wall of brown and red seaweeds, respectively, and a thermal gelation process. The agarose/fucoidan (AgaFu) blended hydrogels were obtained by dissolving Aga in 3 or 5 wt % Fu aqueous solutions to obtain different proportions (4:10; 5:10, and 7:10 wt). The rheological tests on hydrogels revealed a non-Newtonian and viscoelastic behavior, while the characterization confirmed the presence of the two polymers in the structure of the hydrogels. In addition, the mechanical behavior showed that increasing Aga concentrations resulted in hydrogels with higher Young's modulus. Further, the ability of the developed materials to sustain the viability of human pancreatic cells was assessed by encapsulation of the 1.1B4HP cell line for up to 7 days. The biological assessment of the hydrogels revealed that cultured pancreatic beta cells tended to self-organize and form pseudo-islets during the period studied.

Topics: Diabetes Mellitus; Humans; Hydrogels; Polysaccharides; Sepharose

Use of hemoglobin A1c point-of-care devices in physician offices provides immediate results and reduces inconveniences for the patients. We compared the analytical performances of 3 point-of-care HbA1c analyzers to high pressure liquid chromatography (HPLC).. We preselected a pool of 40 EDTA-preserved whole blood samples from our laboratory with HbA1c results obtained by HPLC (mean 6.6% [49 mmol/mol] and range: 4.6-9.9% [27-87 mmol/mol]). Aliquots of theses samples were tested by Afinion AS100, DCA Vantage and In2it point-of-care systems. According the Clinical Laboratory Standards Institute EP-09 protocol we determined linearity (linear regression and correlation coefficient between point-of-care and reference methods), bias (Bland-Altman analysis) and coefficient of variation (%). We used the acceptability criteria endorsed by the National Glycohemoglobin Standardization Program.. The calculated correlation coefficients (r) were 0.98, 0.98 and 0.83 for Afinion AS100, DCA Vantage and In2it systems, respectively. The 95% confidence interval of the error between point-of-care systems and the reference method was -0.41% and +0.34% (p =.22) for Afinion AS100, -0.62% and +0.05% (p =.57) for DCA Vantage, and -1.15% and +1.26% (p<.001) for the In2it. The coefficients of variation for Afinion AS100, DCA Vantage and In2it systems were 1.80, 3.74 and 7.14%, respectively.. Only the Afinion AS100 point-of-care system met all NGSP performance criteria.

Topics: Chromatography, Affinity; Chromatography, High Pressure Liquid; Data Accuracy; Diabetes Mellitus; Glycated Hemoglobin; Humans; Latex Fixation Tests; Linear Models; Point-of-Care Testing; Reproducibility of Results; Sepharose; Statistics, Nonparametric

Tungsten disulfide (WS2) nanosheets were discovered to possess intrinsic peroxidase-like activity and catalyze the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) to produce a color reaction in the presence of H2O2. Based on this finding, a colorimetric method and a portable test kit for the visual detection of blood glucose have been developed by using glucose oxidase (GOx) and WS2 nanosheets-catalyzed reactions. The linear range for glucose was ranged from 5 to 300 μM (R(2)=0.999) with the detection limit of 2.9 μM. The portable test kit was successfully evaluated glucose levels in serum samples from normal persons and diabetes persons by the observable color change from pale yellow to yellow-green, blue-green.

Topics: Biosensing Techniques; Blood Chemical Analysis; Blood Glucose; Colorimetry; Diabetes Mellitus; Glucose Oxidase; Humans; Hydrogen Peroxide; Metal Nanoparticles; Sepharose; Tungsten Compounds

Molybdenum disulfide (MoS2) has attracted increasing research interest recently due to its unique physical, optical and electrical properties, correlated with its 2D ultrathin atomic-layered structure. Until now, however, great efforts have focused on its applications such as lithium ion batteries, transistors, and hydrogen evolution reactions. Herein, for the first time, MoS2 nanosheets are discovered to possess an intrinsic peroxidase-like activity and can catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. The catalytic activity follows the typical Michaelis-Menten kinetics and is dependent on temperature, pH, H2O2 concentration, and reaction time. Based on this finding, a highly sensitive and selective colorimetric method for H2O2 and glucose detection is developed and applied to detect glucose in serum samples. Moreover, a simple, inexpensive, instrument-free and portable test kit for the visual detection of glucose in normal and diabetic serum samples is constructed by utilizing agarose hydrogel as a visual detection platform.

Topics: Benzidines; Blood Glucose; Colorimetry; Diabetes Mellitus; Disulfides; Dose-Response Relationship, Drug; Humans; Hydrogels; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Microscopy, Electron, Transmission; Molybdenum; Nanostructures; Nanotechnology; Peroxidase; Sepharose; Temperature

Islet xenografts from porcine donors can reverse diabetes in experimental animal models and may be an alternative to human islet transplantation. We have recently reported the ability of porcine islets encapsulated in a double layer of hydrophilic agarose to maintain in vitro functional ability for >6 months. Although beta-cells are capable of adapting their secretory capacity in response to glucose levels, evidence has shown that prolonged hyperglycemia can compromise this ability. The aim of the present study was to determine the effects of diet manipulation on the long-term regulation of blood glucose levels, and the preservation of functional islet in the macrobeads. Twenty-one streptozotocin-induced diabetic Wistar-Furth male rats were randomly assigned to two diets containing 64% carbohydrate (CHO) or 20% CHO. Groups of five to six animals assigned to either diet were implanted with either empty (EM) or porcine islet-containing macrobeads (PIM) and followed for 333 days. Observations included general condition, body weight, blood glucose, and food and water intakes. Monthly blood samples were collected for insulin and C-peptide analysis. The 20% CHO diet significantly lowered blood glucose values when compared with those of the 64% CHO groups for both the empty (14.94 +/- 0.41 vs. 16.26 +/- 0.42 mmol/L, respectively, p < 0.001) and islet macrobead recipients (12.88 +/- 0.39 vs. 15.57 +/-0.85 mmol/L, respectively, p <0.001). The different diets, however, had no statistically significant effects on the preservation of islet mass in the macrobead. Serum porcine C-peptide was detected throughout the experiment in animals receiving porcine islet macrobeads, regardless of diet. Diabetic rats fed a low carbohydrate level diet and transplanted with porcine islet macrobeads had improved total tissue glucose disposal and improved clinical parameters associated with diabetes.

Topics: Animals; Blood Glucose; Capsules; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diet, Carbohydrate-Restricted; Glucose Tolerance Test; Humans; Insulin; Islets of Langerhans Transplantation; Male; Rats; Rats, Inbred BB; Rats, Inbred WF; Sepharose; Swine; Transplantation, Heterologous

In this study, the insulin secretory characteristics of the microencapsulated hamster islets were studied during long-term culture. The hamster islets were encapsulated as single-layer agarose microbeads or three-layer agarose microbeads with agarose and agarose containing poly(styrene sulfonic acid) (PSSa), respectively. The influence of PSSa on the function of the rat islets microencapsulted in three-layer microbeads was primarily monitored. The aim of this study was to examine the influence of the PSSa on the in vitro function of the islets encapsulated in the agarose/PSSa microbeads compared with single-layer agarose microbeads during long-term culture. The microbeads were cultured for 30 days in medium of Eagle's MEM at 37 degrees C in 5% CO2 and 95% air. The basal insulin secretion into the culture medium was measured daily during the first 12 days and two times per week until 30 days. The microbeads were subjected to static incubation test on the 10th, 20th, and 30th day during culture. The basal insulin secretion level of the agarose/PSSa microbeads was significantly higher than that of single-layer agarose microbeads. The static incubation tests revealed a similar pattern of insulin secretion from both microbeads when they were exposed to high glucose challenge. In the static incubation test, both could significantly increase insulin release to more than 6.61 times (stimulation index) in response to high glucose stimulation and could significantly decrease when glucose concentration returned from high glucose to low glucose on the 10th, 20th, and 30th day of culture. This study demonstrated that the hamster islets enclosed in agarose/PSSa hydrogel not only continuously secreted basal amounts of insulin, but also maintained their response to high glucose stimulation similar to the agarose microbeads. The above results together with those of our previous in vivo study suggest that the three-layer microbeads (agarose/PSSa) are well suitable for xenotransplantation of islets for the clinical application.

Topics: Animals; Cricetinae; Culture Techniques; Diabetes Mellitus; Humans; Hydrogels; Insulin; Insulin Secretion; Islets of Langerhans; Male; Microspheres; Pancreas, Artificial; Particle Size; Polystyrenes; Sepharose; Time Factors

A fundamental cause of diabetic microalbuminuria, heterogeneity of normal and diabetic urinary albumin was shown by affinity chromatography on Cibacron Blue F3GA. By changing the properties of interaction with the matrix, the protein was separated into six fractions. Samples of urinary albumin from proteinuria patients showed the same elution profiles as those of serum albumin, whereas those from controls or normoalbuminuria diabetic patients exhibited different elution patterns. The relative percentage of the resin unbound fraction of urinary albumin was ten or more times higher than that of serum albumin, and the ratio decreased with increasing albumin excretion into urine. More than 6 mol fatty acid/mol albumin combined with the unbound fraction. It is suggested that microalbumin excretion into urine is the result of excessive unesterified fatty acid binding to the protein.

Topics: Adult; Albumins; Albuminuria; Chromatography, Affinity; Coloring Agents; Diabetes Mellitus; Humans; Middle Aged; Sepharose

Simple techniques for measurement of glycosylated hemoglobin and glycosylated albumin by affinity chromatography on m-aminophenylboronic acid agarose columns have recently been developed. This study explored the time course of changes in glycoalbumin versus those of glycohemoglobin in response to rapid changes in ambient glucose concentration. One would predict that glycoalbumin levels would change more rapidly than glycohemoglobin levels due to the shorter half-life of albumin than hemoglobin. This was found to be the case in a group of rabbits rendered diabetic with alloxan. Glycoalbumin levels plateaued 4 weeks after alloxan administration, while glycohemoglobin levels were still rising. In a group of diabetic patients in whom glucose levels were initially poorly controlled, strict diet or intensive insulin management were used to rapidly bring glucose levels under control. In this group of patients, the glycoalbumin values entered the normal range and plateaued, while glycosylated hemoglobin levels were still falling. Glycoalbumin determination by affinity chromatography is a valuable adjunct to glycosylated hemoglobin determination in evaluating near term control of blood sugar values.

Topics: Animals; Blood Glucose; Chromatography, Affinity; Chromatography, Agarose; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diet, Diabetic; Glycated Hemoglobin; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Hypoglycemic Agents; Rabbits; Sepharose; Serum Albumin; Time Factors

The determination of glycosylated albumin was performed in 19 diabetic patients and in 16 control subjects, by means of chromatographic separation on columns of phenilboronic acid, immobilized by agarose gel. The interference with free glucose was eliminated by serum gel filtration on sephadex G 25 columns. The results demonstrated that glycosylated albumin values, obtained by affinity chromatography, discriminate diabetic by non-diabetic subjects and significantly correlate with glycosylated proteins levels, taken by colorimetric method with thiobarbituric acid. The chromatographic technique resulted in a more simplified way than colorimetric method and has shown to be sensitive to significant increases in "in vitro" glycosylation. Moreover it lasts less than the other technique and its variation coefficient is extremely low. In conclusion it represents a progress in the routine determination of glycosylated albumin.

Topics: Adult; Aged; Chromatography, Affinity; Colorimetry; Diabetes Mellitus; Female; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Male; Middle Aged; Sepharose; Serum Albumin

We evaluated the effect of azotemia on results for glycated hemoglobin as measured by a boronate-agarose affinity method and an ion-exchange chromatographic procedure with saline preincubation and found a good correlation. However, values for glycated hemoglobin in samples from nondiabetic patients with various degrees of azotemia were consistently higher with the ion-exchange column procedure (mean, 8.5%) than with the boronate affinity method (mean, 6.2%). The latter method may thus be preferred for monitoring glycated hemoglobin in diabetic patients with impaired renal function.

Topics: Boronic Acids; Chromatography, Affinity; Chromatography, Ion Exchange; Diabetes Mellitus; False Positive Reactions; Gels; Glycated Hemoglobin; Humans; Sepharose; Uremia

Glucosylated albumin of human serum isolated by dye-ligand chromatography on blue Sepharose, was not found to be completely reducible by sodium borohydride. The percentage reducible hexose as judged by phenol-sulphuric acid reaction was in the range of 49.7 +/- 12.8 in control subjects (n = 24) and 53.8 +/- 14.2 in diabetics (n = 50). Increase in the level of total hexose bound to albumin and reducible hexose were equally significant in diabetes (P less than 0.001). Sodium chloride gradient elution during chromatography on blue Sepharose showed that glucosylated albumin had lesser affinity than the native protein to the matrix. It is proposed that an addition product between hexose and albumin is formed during nonenzymatic reaction and this adduct is fairly stable and is not reducible by sodium borohydride.

Topics: Adult; Blood Glucose; Borohydrides; Chromatography, Agarose; Diabetes Mellitus; Hemoglobins; Hexoses; Humans; Middle Aged; Oxidation-Reduction; Sepharose; Serum Albumin

A new procedure has been devised for the isolation of a glycosyl subunit of albumin from human serum. After the selective removal from serum proteins with affinity chromatography on Blue-Sepharose CL-6B, albumin was applied to a column of concanavalin-A Sepharose which resolved the protein in two subunits with different specific colour activity for carbohydrates, as tested with thiobarbituric acid assay. The glycosyl albumin bound to concanavalin-A Sepharose was homogeneous when examined by immunoelectrophoresis and sodium dodecyl-sulphate polyacrylamide electrophoresis, whereas it showed a microheterogeneity when tested by isoelectric focusing. The procedure was applied to a model system as well as to serum from normal and diabetic patients.

Topics: Carbohydrates; Chemical Phenomena; Chemistry; Chromatography; Diabetes Mellitus; Electrophoresis, Polyacrylamide Gel; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Immunoelectrophoresis; Isoelectric Focusing; Sepharose; Serum Albumin

The use of m-aminophenylboronic acid immobilized on agarose as the affinity matrix for the separation and quantitation of Glyco Hb has been investigated. The glyco fraction isolated from the affinity columns contained about 10% Hb A1a + b, 52% Hb A1c, and 38% Hb A0-like glyco components. The nonglyco fraction had major portions of Hb A1a + b and Hb A0 and a small amount of Hb A1c (also contained Hb F). In normals and diabetic patients, the comparison of the affinity method with the ion-exchange chromatographic, fluorometric, and colorimetric methods showed good correlation. The affinity method also showed acceptable precision and reproducibility. The presence of labile aldimine did not influence the affinity method because it seems that only the stable ketoamine was bound to the affinity gel and thus separated by this method. Moreover, this method was less sensitive to variations in column temperature and sample age. Glyco Hb levels were determined in newborn infants and in adults with various hemoglobinopathies. The results indicated that the presence of Hb F, Hb S, and Hb C did not affect the glyco Hb determinations. Thus the affinity chromatographic approach has wider application than ion-exchange chromatography and is, at the same time, much simpler and faster than the chemical methods.

Topics: Adult; Boronic Acids; Chromatography, Affinity; Chromatography, Ion Exchange; Colorimetry; Diabetes Mellitus; Female; Fluorometry; Glycated Hemoglobin; Hemoglobinopathies; Humans; Imines; Infant, Newborn; Male; Sepharose

Topics: Antibodies; Antigen-Antibody Complex; Antigens; Chemical Precipitation; Chromatography, Affinity; Chromatography, Gel; Diabetes Mellitus; Humans; Insulin Antibodies; Polyethylene Glycols; Rheumatoid Factor; Sepharose; Staphylococcal Protein A