Advanced glycation end products as a source of artifacts in immunoenzymatic methods

Aleksandra Kuzan,A Gamian,K Maksymowicz,C Pezowicz,A Bronowicka-Szydełko,K Stach,A Chwiłkowska

doi:10.1007/s10719-017-9805-4

Abstract

The most abundant proteins in the arteries are those of extracellular matrix, ie. collagen and elastin. Due to their long half-lifes these proteins have an increased chance to undergo glycation. The aim of this study was to determine relationship between the content of the main extracellular matrix proteins and the advanced glycation end products (AGEs) in arteries. In this study 103 fragments of aorta were analyzed by ELISA and immunobloting for the content of collagens type I, III and IV and elastin and the content of advanced glycation end-products (AGE). A negative correlation between the content of collagens type III and IV and AGE (r = −0,258, p = 0,0122, and a weak negative correlation between collagen type III and age of the sample donor (r = 0,218, p = 0,0262) were demonstrated. This result comes as a surprise and it contradicts an intuitive assumption that with more glycation substrate, i.e. matrix proteins, more AGE products are expected. We have concluded that the results of the ELISA tests must have been influenced by the glycation. As a consequence, either modified protein molecules were not being recognized by the antibodies, or the glycation, and formation of crosslinks have blocked access of the antibodies to the antigen. It will conceal the effect of the linear dependence between the result (absorbance/densitometry) from the quantity of protein to which the antibody is directed.

Highlights

Glycation is a non-enzymatic process, where reducing sugars react with amino groups of proteins
ELISA analysis revealed that the artery wall contains on average of 19.38 (+/− 11.74) mg/g of tissue collagen, 15.85 (+/− 10.12) mg/g of tissue elastin and 0.522 (+/− 0.373) mg/g tissue of the AGE antigen
There has been no significant correlation between the contents of the advanced glycation end-products and the content of collagen type I, the sum of collagens and elastin

Summary

Introduction

Glycation is a non-enzymatic process, where reducing sugars (glucose, fructose, glucose-6-phosphate and other) react with amino groups of proteins. It occurs spontaneously inside and outside of living organisms. It is known that they constitute over 50% of the dry weight of the artery [5] and that the quantity of this tissue may be modulated by different processes, for example atherogenesis Still, it is unclear whether the amount of collagen and elastin grows, or Glycoconj J (2018) 35:95–103 falls with age. We have previously described the relationship between collagen type II and the degree of atherosclerosis [11] and among other types of collagen and degree of disease (unpublished data) This is work where our focus is on the products of advanced glycation associated with extracellular matrix proteins

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Discussion

Conclusion