sodium-borohydride and Diabetes-Mellitus

Nitric oxide (NO) donors sodium nitrosoprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonemine (SIN-1) caused a time- and concentration-dependent loss of catalytic activity of recombinant human placental aldose reductase. Modification of the enzyme was prevented by NADPH and NADP and reversed partially by dithiothreitol (DTT) and sodium borohydride. The protection by NADPH was lost in the presence of both substrates (NADPH and glyceraldehyde), indicating that the enzyme becomes sensitive to inhibition by SNP during catalysis. Site-directed mutant form of the enzyme, in which active site cys-298 was substituted with serine (C298S) was not inactivated by NO donors, whereas, ARC80S and ARC303 were as sensitive as the wild type enzyme, indicating that inactivation of aldose reductase is due to modification of the active site at cys298. These results suggest that NO may be an endogenous regulator of aldose reductase, and consequently the polyol pathway of glucose metabolism; which has been implicated in the pathogenesis of secondary diabetic complications.

Topics: Aldehyde Reductase; Binding Sites; Borohydrides; Catalysis; Diabetes Complications; Diabetes Mellitus; Dithiothreitol; Enzyme Inhibitors; Glyceraldehyde; Humans; Kinetics; Molsidomine; Mutagenesis, Site-Directed; NADP; Nitric Oxide; Nitroprusside; Penicillamine; Placenta; Recombinant Proteins; S-Nitroso-N-Acetylpenicillamine

The chemistry of the fructosamine assay was studied by using the Amadori compound, N alpha-formyl-N epsilon-fructose-lysine (fFL), an analog of glycated lysine residues in protein. Previously (Clin Chem 1993;39:2460-5), we reported that free lysine was formed from fFL at 70% yield during incubation with alkaline nitroblue tetrazolium (NBT) under the conditions routinely used for the fructosamine assay (sodium carbonate buffer, pH 10.35 at 37 degrees C). Here, we show that D-glucosone is the primary carbohydrate oxidation product formed from Amadori compounds in the fructosamine assay. Glucosone, which decomposes under alkaline assay conditions with a half-life of < 30 min, reaches a maximum concentration of approximately 50% of the initial fFL concentration after 10 min of incubation. Like fFL, glucosone reduces NBT to the purple monoformazan dye, but its decomposition is not accelerated by the presence of NBT. The dicarbonyl-trapping reagent, aminoguanidine, inhibits the fructosamine assay by approximately 25% when fFL is the substrate, but by nearly 100% with glucosone as substrate. Studies with serum samples from diabetics and nondiabetics indicate that glucosone formation does not have a significant effect on the clinical usefulness of the fructosamine assay; however, corrections for glucosone formation may be required when the assay is used for estimating the extent of glycation of proteins.

Topics: Borohydrides; Diabetes Mellitus; Fructosamine; Gas Chromatography-Mass Spectrometry; Hexosamines; Humans; Ketoses; Kinetics; Lysine; Nitroblue Tetrazolium; Oxidation-Reduction; Reference Values

1. A radiochemical assay has been developed to measure non-enzymic glycosylation (glycation), which is free from interference from enzymic glycosylation and which can be used in batch analysis. 2. Glycated residues were isolated by affinity chromatography on mini-columns, after reduction with Na[3H]BH4 and hydrolysis. 3. The specific activity of the labelled borohydride was measured by a novel procedure. 4. The method was used to measure glycation of albumin, immunoglobulin G and fibrinogen isolated from diabetic and non-diabetic subjects.

Topics: Borohydrides; Chromatography, Affinity; Diabetes Mellitus; Electrophoresis, Polyacrylamide Gel; Fibrinogen; Glycoproteins; Glycosylation; Humans; Hydrolysis; Immunoglobulin G; NAD; Oxidation-Reduction; Radiochemistry; Serum Albumin

We studied 11 diabetic patients, all of whom had severe atherothrombotic disease, and 11 normal controls. Overall glycation was assessed by the extent of incorporation of [3H]-NaBH4 into fructosyl lysine separated from whole platelet proteins following aminoacid analysis. Fructosyl lysine represented 5.7% +/- 1.0 S.D. of the total radioactivity in the normal whole platelet samples. Increased glycation was observed in platelets from 5 of the 11 diabetics. Platelet glycation did not correlate with glycation of hemoglobin or albumin. The pattern of glycation of various platelet proteins in whole platelets, as determined by the incorporation of [3H]-NaBH4 into electrophoretically separated proteins did not display selectivity, although myosin and glycoproteins IIb and IIIa showed relatively increased levels of [3H]-NaBH4 incorporation. Artificially glycated platelet membranes exhibited glycation mainly in proteins corresponding to the electrophoretic mobility of myosin, glycoproteins IIb and IIIa.

Topics: Amino Acids; Blood Platelets; Blood Proteins; Borohydrides; Diabetes Mellitus; Glycated Hemoglobin; Glycated Serum Albumin; Glycation End Products, Advanced; Glycosylation; Humans; Hydrolysis; Lysine; Serum Albumin

Topics: Borohydrides; Diabetes Mellitus; Diabetic Ketoacidosis; Glycation End Products, Advanced; Humans; Lipoproteins, VLDL; Serum Albumin, Bovine

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