Analysis of advanced glycation end product (AGE) content in tendon tissue with fluorometric and colorimetric assays

Abstract

Being the most abundant protein in the human body, constituting about ~ 25 % of all protein mass, collagen provides the necessary biomechanical characteristics, structural stability, and protection to tissues. Out of 28 up to now discovered collagens, type I is the most abundant. It is found in skin and load-bearing tissues such as bone, tendons, and ligaments. Due to its importance, the insight into mechanisms influencing the changes in collagen type I during ageing, disease and injuries are worthwhile investigating.Collagen biosynthesis and further assembly is a complex multistep process, from forming a peptide chain, to triple helix assembly and crosslinking of collagen molecules, which is necessary to ensure the required sturdy structure of tissues. Crosslinking is triggered enzymatically and non-enzymatically, with the enzymatic crosslinking resulting in desired immature and mature inter- and intramolecular crosslinks. Their formation enhances the strength of collagen-formed tissues.As opposed to enzymatic crosslinking process, the formation of non-enzymatic crosslinks increases with ageing and disease. The products of non-enzymatic crosslinking are the so-called advanced glycation end products (AGEs), originating from an intricate cascade of modification processes, known as glycation. During various glycation pathways, sugars attach to proteins or lipids, especially affecting long-lived and abundant proteins such as collagen. They accumulate irreversibly, eventually compromising functionality and reducing healing capacity. AGEs have been, besides ageing, linked to various degenerative diseases such as diabetes, cardiovascular diseases, osteoarthritis, and Alzheimer’s, making the determination of their content and influence on tissues important.Within this work, an experimental method for the determination of total fluorescent AGEs (fAGEs) per tendon tissue hydroxyproline content using a microplate reader was established, following known protocols for bone from other laboratories.The protocol used for the quantification of the total content of fAGEs in tendon tissue hydrolysates is comprised of two parts - a fluorometric assay employing quinine as a fluorescence standard for quantification of the bulk content of AGEs and a colorimetric assay determining the content of hydroxyproline in tissue samples. Hydroxyproline is a collagen-specific amino acid, used as an indicator of tissue collagen content. The amount of fluorescent AGEs is then reported in nanograms of quinine per milligram of hydroxyproline.For the purpose of method validation, test samples were created by an in-vitro crosslinking method inducing the formation of AGEs. Some of these AGEs exhibit fluorescence, making them measurable with a fluorometric assay.Sample tissue used were the tail tendons of 18-month-old male BALB/c mice divided into control and artificially non-enzymatically crosslinked groups. For induction of in vitro crosslinking, methylglyoxal (MGO) was used. MGO is a highly reactive α-dicarbonyl compound, a by-product of the glycolysis pathway, present in vivo as one of the physiological inducers of AGEs. MGO-treated samples are expected to result in a higher amount of total fAGEs per hydroxyproline.The artificial glycation effect was detectable already during visual inspection of the samples after treatment, causing browning of the MGO-treated tissue. In repeated assays, MGO treated samples consistently resulted in a higher fAGE content compared to the control buffer-treated sample group, validating the measurement method.