The molecular weights of plasma proteins from healthy subjects and from patients with well-or badly-controlled diabetes mellitus have been determined by use of a matrix-assisted laser desorption ionization method, representing a highly accurate technique for the determination of the molecular weight of large biomolecules. Using this approach, different molecular weights of human serum albumin have been found for healthy (66,572-66,694 dalton) and diabetic (66,785-68,959 dalton) subjects. Such differences can be rationalized as being due to the different number of glucose molecules condensed on the protein and/or their further oxidation products; in the case of our diabetic patients this number is in the range of 1.4-14.8. The data show the high validity and specificity of the technique, which allows us to evaluate, without any protein degradation procedure, the number of glucose molecules condensed on a specific protein and ascertain the relationship of this number to the physiopathogenetic conditions of the subjects studied.
The products arising from the reactions of ribonuclease with glucose or fructose have been studied by means of matrix-assisted laser desorption/ionization mass spectrometry. The reactions have been carried out at physiological pH, with two different sugar concentrations and different incubation times. A maximum increase in molecular weight, corresponding to 485 Da, was found in the case of ribonuclease incubated with 0.25 M fructose for 6 days. Furthermore clear differences have been found in the reactivity of glucose and fructose; in particular, while glucose reacts faster than fructose in the early stage of the glycation, the rearrangements of the Amadori adducts are favoured using fructose as reagent sugar.
A new method, based on matrix-assisted laser desorption/ionization (MALDI) measurements, has been developed for the evaluation of the glycation level of globins, a relevant parameter for diabetes control. It shows high levels of reproducibility and specificity, allowing the different glycated and glyco-oxidized products of both alpha- and beta-globins to be distinguished. Such specificity is reflected in its possible diagnostic use not only for the control of diabetes, but also for the occurrence of 'oxidative stress'. The comparison, for the same samples, of the obtained MALDI data with the related HbA1c values, determined by a high performance liquid chromatographic method, allowed it to be established that HbA1c percentages are not, as usually retained, related to the simply glycated beta-globin, but to the whole pool of glycated and glyco-oxidized alpha- and beta-globins.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been employed for the evaluation of the glycation level of IgG from healthy subjects and also from well- and badly-controlled diabetic patients. The measurements have been performed on untreated plasma protein fractions. The data obtained have shown that a clear mass increase, originating from non-enzymatic glycation processes, is observed in the case of diabetic patients: for well-controlled ones it is in the range 512-1565 Da, while it becomes 827-4270 Da for badly-controlled diabetic patients. This approach indicates that MALDI mass spectrometry is a highly specific tool that can be employed in the metabolic control of diabetic patients and in studies relating the IgG glycation level to possible immunological impairment.
Matrix-assisted laser desorption/ionization (MALDI) has been employed for the determination of molecular weights of alpha- and beta-globins obtained from blood samples of healthy and diabetic subjects. Glycated species, originating from the reaction of glucose with the proteins, are easily evident. The MALDI measurements allowed one to distinguish between simple glycation and glyco-oxidation processes of haemoglobin. In fact, together with the products arising from simple glucose condensation on the two proteins, compounds at different oxidation levels were evident. Due to its high specificity, MALDI must be considered a valuable tool for diagnosis in the diabetes field.
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