RESUMO
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.
Assuntos
Globinas/química , Glucose/química , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Globinas/isolamento & purificação , Hemoglobinas Glicadas/análise , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Imunoglobulina G/química , Idoso , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Glucose/química , Glucose/metabolismo , Humanos , Pessoa de Meia-Idade , Albumina Sérica/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Globinas/química , Diabetes Mellitus/sangue , Glucose/química , Glicoproteínas/química , Hemoglobinas/química , Humanos , Técnicas In Vitro , Pessoa de Meia-Idade , Peso Molecular , Oxirredução , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The in vitro reactions of RNase with different concentrations of glucose or fructose have been studied by means of electrospray mass spectrometry coupled with microcolumn high performance liquid chromatography. The results obtained have shown that, subsequent to the protein glycation, a series of cross-linking products are generated. Their molecular weights demonstrate that severe degradation processes of the proteic substrate takes place after the cross-linking process.
Assuntos
Ribonucleases/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Glucose/química , Glucose/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Ribonucleases/químicaRESUMO
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.
Assuntos
Glucose/química , Muramidase/química , Soluções Tampão , Reagentes de Ligações Cruzadas , Frutose/química , Fosfatos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
