sodium-dodecyl-sulfate and Diabetes-Mellitus

Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.

Topics: Blotting, Western; Cell Line; Diabetes Mellitus; Ghrelin; Glucagon-Like Peptide 1; Humans; Insulin; Pancreatic Polypeptide; Peptide Hormones; Proinsulin; Protein Precursors; Sodium Dodecyl Sulfate; Somatostatin

An analytical method has been developed for the separation of glyoxal (Go), methylglyoxal (MGo), and dimethylglyoxal (DMGo) by MEKC using stilbenediamine (SD) as derivatizing reagent, separation time 6.5 min, SDS as micellar medium at pH 8, and sodium tetraborate (0.1 M) as buffer. Uncoated fused-silica capillary, effective length 50 cm x 75 microm id; applied voltage 20 kV and photodiode array detection, were used. Calibration was linear within 0.02-150 microg/mL with detection limits 3.5-5.8 ng/mL. Go and MGo, observed for diabetic and healthy volunteers, were within 0.098-0.193 microg/mL Go and 0.106-0.245 microg/mL MGo with RSD 1.6-3.5 and 1.7-3.4%, respectively, in diabetics against 0.016-0.046 microg/mL Go and 0.021-0.066 microg/mL MGo with RSDs 1.5-3.5 and 1.4-3.6%, respectively, in healthy volunteers. Go and MGo in diabetics were also measured by standard addition and DMGo as an internal standard. Additives do not contribute significantly to Go and MGo matrix.

Topics: Calibration; Chromatography, Micellar Electrokinetic Capillary; Diabetes Mellitus; Glyoxal; Humans; Pyruvaldehyde; Reproducibility of Results; Sensitivity and Specificity; Sodium Dodecyl Sulfate; Stilbenes

The lipoproteins of rats fed a high sucrose diet and made diabetic by administration of 45 mg/kg of streptozotocin were studied. All lipoprotein classes were found to be present in increased concentrations. The apolipoprotein composition of the various lipoprotein fractions was studied by polyacrylamide-gel electrophoresis in the presence of 8 M urea, isoelectric focusing in the presence of 8 M urea, and sodium dodecyl sulfate gel electrophoresis in polyacrylamide gels. In the very low density lipoproteins (VLDL) of diabetic rats, there was a marked alteration in the relative amounts of C proteins by polyacrylamide-gel electrophoresis, and this was found by isoelectric focusing to be primarily a relative increase in C-III-3 apoprotein and a decrease in C-III-O. In addition, in the diabetic rats, the VLDL contained a protein of mol wt 46,000, the A-IV protein, which normally is only present in the high density lipoproteins. In the high density lipoproteins, (HDL) the same alterations in pattern of the C proteins seen in the VLDL were present. Furthermore, the arginine-rich and A-IV protein normally present in HDL could not be detected in the HDL, although the other apolipoproteins are present. Apolipoprotein concentrations were determined by quantitative immunoelectrophoresis. It was found that in the diabetic rats there was an increase in the total amount of apo-B in the plasma, with the increment divided proportionately between the VLDL and the low density lipoprotein (LDL). The total apo-C concentration of plasma increased minimally. The A-IV concentration of plasma increased by 27%; it decreased markedly in the HDL, but appeared in increased amounts in both VLDL and in the d greater than 1.21 fraction. The arginine-rich protein decreased by 63% in the plasma and decreased significantly in the HDL, but increased in VLDL, LDL, and in the d greater than 1.21 fraction. These alterations in apolipoprotein patterns in diabetic animals suggest that the apolipoproteins may play an important role in determining the concentration of various lipoprotein fractions, or may be the result of altered metabolism of the lipoproteins. These lipoproteins with altered apolipoprotein composition may have important biologic differences from normal lipoporteins. Nevertheless, the HDL, despite the fact that it is deficient in some of its major constituents, was unchanged in its cholesterol content.

Topics: Animals; Apoproteins; Diabetes Mellitus; Electrophoresis, Polyacrylamide Gel; Immunoelectrophoresis; Isoelectric Focusing; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Lipoproteins, VLDL; Male; Rats; Sodium Dodecyl Sulfate; Streptozocin; Sucrose

Topics: Adolescent; Adult; Aged; Child; Diabetes Mellitus; Electrophoresis, Polyacrylamide Gel; Female; Humans; Hypertension; Kidney Diseases; Kidney Glomerulus; Kidney Tubules; Male; Methods; Middle Aged; Molecular Weight; Nephrotic Syndrome; Proteinuria; Sodium Dodecyl Sulfate; Urine

Topics: Animals; Cellulose; Chromatography, Gel; Diabetes Mellitus; Dialysis; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Evaluation Studies as Topic; Horses; Humans; Hydrogen-Ion Concentration; Hypertension; Immunoelectrophoresis; Kidney Diseases; Methods; Molecular Weight; Proteinuria; Sodium Dodecyl Sulfate; Time Factors; Ultrafiltration