Clinical studies on α1-acid glycoprotein glycosylation - with focus on fucose

University dissertation from Linköping : Linköpings universitet

Abstract: α1-Acid glycoprotein (AGP, orosomucoid) is a heavily glycosylated protein found in blood plasma in all humans. AGP is one of the major acute phase proteins, and its concentration is frequently measured in clinical laboratories as a marker for inflammatory disease. For several years it has been known that the glycosylation of AGP is altered in different physiological and pathological conditions. However, methods for analysis of the glycosylation changes have been time-consuming and at best semi-quantitative. Consequently, clinical studies have been limited to small numbers of patients, and have not confirmed the usefulness of routine analysis of A GP glycosylation in clinical investigations. In the present study, two major issues were addressed: the need for new methods that would allow quantitative measurements of specific glycosylation changes, and the need for clinical studies with sufficient numbers of study subjects to evaluate the potential advantages and drawbacks of analyzing AGP glycosylation in a clinical laboratory.High-pH anion-exchange chromatography (HPAEC) separates oligosaccharides with high resolution and was used to study N-linked oligosaccharides (N-glycans) released from AGP obtained from patients with different inflannnatory conditions. We showed that HPAEC is a very reproducible and useful method for profiling of AGP N-glycans, providing infmmation on N-glycan sialylation and fucosylation simultaneously. In particular, we found a typical pattern with a sharp increase in fucosylated, tri-sialylated N-glycans in patients with rheumatoid arthritis (RA). The laborious preparation of samples prior to analysis is a drawback for the use ofHPAEC as a routine method in a clinical laboratory. However, HPAEC has a great potential as a tool for profiling AGP glycosylation in different pathologic conditions, and it was used as a reference for the development of other analytical methods. In order to analyze a larger number of patient samples, we developed a lectin immunoassay using a fucose specific lectin from the A!euria aurantia mushroom. The lectin immunoassay was well suited for use in a clinical laboratory and was used to study AGP fucosylation in patients with severe bmns, recent onset rheumatoid arthritis (RA), liver disease, and alcohol abuse. In a time study of 10 patients with severe bums, changes in AGP fucosylation showed a typical pattern, without con-elation to acute phase protein synthesis. This confirmed the results of previous studies indicating that glycosylation is regulated independently from protein synthesis in the acute phase response.We studied AGP fucosylation in 131 patients with recent onset RA. In these patients, AGP fucosy lation was increased, but the con elation with disease activity according to a clinical score (DAS28) was not superior to previously used biochemical markers of inflammation. However, in men with RA who had increased AGP fucosylation at the time of diagnosis, the degree of fucosylation conelated to disease progression during the first year following diagnosis. It remains to be confumed whether AGP fucosylation can provide prognostic information for this patient category.When we studied AGP fucosylation in 261 patients investigated for suspected liver disease, fucosylation was heavily increased in patients with histopathological signs of liver cirrhosis. In addition, AGP fucosylation coiTelated to the severity of disease. By calculating an AGP fucosylation index (AGP-Fl, AGP fucosylation/AGP serum concentration), a high diagnostic accuracy was found for liver cinhosis. Even in patients with cinhosis without any symptoms related to liver disease, AGP-FI was significantly higher than in patients with steatosis or chronic hepatitis. When compared with other biochemical markers of liver disease, AGP fucosylation was among the most specific in discriminating liver cin·hosis, and may be useful in clinical investigations of liver disease.We also studied AGP fucosylation in 21 patients with heavy alcohol abuse admitted to hospital for detoxification. fu the 16 male patients that were studied, AGP fucosylation was significantly increased compared to healthy controls. Furthermore, in these men there was a significant con·elation between AGP fucosylation and other biochemical markers previously used for detection of liver cirrhosis. This study should be extended with a larger number of patients, and histopathological examination following liver biopsy, in order to confirm the value of increased AGP fucosylation as an early marker for cirrhosis in this patient categmy.

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