Methods for assaying nonenzymatic glycosylation.

Open University, Oxford Research Unit, England.
Analytical Biochemistry (Impact Factor: 2.58). 01/1989; 175(2):347-60. DOI: 10.1016/0003-2697(88)90558-1
Source: PubMed
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    ABSTRACT: 5-(Hydroxymethyl)-2-furfuraldehyde (HMF) is formed upon heat treatment of milk and milk-resembling systems by the Maillard reaction, via its Amadori product, and also by isomerization and subsequent degradation of sugars. Traditionally, the HMF content has been used as an indicator of both degradation routes. A new analytical approach has been developed for determining the HMF formed only by the acidic degradation of Amadori products, called bound HMF, and that could be related to the extent of the Maillard reaction due to heat treatment or long-term storage of foods. Optimal conditions for the acidic digestion procedure were determined. Reversed-phase HPLC is applied for accurate measurement of bound HMF.
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    ABSTRACT: To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300: 1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.
    Pharmaceutical Research 04/1999; 16(3):350-8. · 4.74 Impact Factor
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    ABSTRACT: We optimally solubilized the diformazan that forms during the fructosamine reaction with a nonionic detergent; we then automated the assay. After optimal solubilization of diformazan, we found that serum urate contributed significantly to the color formation in fructosamine reaction. When the error introduced by urate was corrected mathematically by use of an experimental determined factor, the serum fructosamine concentrations obtained were comparable with those of a more specific test of the nonenzymatic glycation (HPLC of hydrolyzed proteins). We found that concentrations of fructosamine in serum increased with increasing age of subjects. In diabetic patients the average concentration of fructosamine in serum exceeded that in nondiabetic subjects and correlated with the preprandial serum glucose and with other measures of deteriorating glycemic control (e.g., increased need for insulin therapy). Serum fructosamine normalized for albumin content was also increased in patients with chronic renal failure. Multiple mechanisms (including exposure of patients to hypertonic glucose during dialysis) appear to be involved in the increase of serum fructosamine in patients with chronic renal failure.
    Clinical Chemistry 11/1990; 36(10):1825-30. · 7.15 Impact Factor