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.

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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.

The non-enzymatic reactions between glucose or fructose with lysozyme, performed under pseudo-physiological conditions, have been studied by means of matrix-assisted laser desorption/ionization mass spectrometry. Phosphate buffer at concentrations of 0.05, 0.2 and 0.5 M has been employed. The formation of glycated proteins as well as of cross-linking products has been always observed. In the case of glucose, high phosphate buffer concentrations affect the glycation kinetics and promote the formation of cross-linking products. With fructose, such influence is moderate, the reaction kinetics being mainly influenced by the higher reactivity of the sugar.

Pyrolysis/gas chromatography/mass spectrometry was applied to the study of products arising from the interaction between glucose and poly-L-lysine. The comparison of pyrograms of control poly-L-lysine and glycated poly-Llysine ied to the identitication of six different compounds, four of which were furan derivatives that can be considered as markers for advanced glycation processes.

The nature of the products arising from a 10 days, sterile incubation at 37°C and pH 7.2 of a 1:1 mixture of N-α-(p-tosyl)-lysine-methylesterhydrochloride and anhydrous D-glucose was investigated by fast atom bombardment mass spectrometry and(1)H and(13)C nuclear magnetic resonance spectroscopies. Differently to the reactivity usually described on the basis of other analytical techniques, FAB mass spectrometric measurements indicate the occurrence of the reaction of protected lysine with more than one D-glucose molecule.

The structural investigation of the products arising from 28 days incubation of albumin with high glucose concentration and further enzymatic hydrolysis has been carried out by means of high-performance liquid chromatography/mass spectrometry (HPLC/MS) under plasmaspray conditions. By this approach many different compounds have been detected, and for most of them, possible structures have been proposed on the basis of literature data and molecular weight assignments.

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