RADIORECEPTOR ASSAY FOR ADVANCED GLYCOSYLATION END-PRODUCTS

Previous assays for nonenzymatic advanced glycosylation end products (AGEs) formed in tissues and/or circulating in blood are unsatisfactory. Based on our earlier identification of AGE-specific receptors on the macrophagelike tumor cell line RAW 264.7, a new assay system for AGEs has been devised. RAW 264.7 cells were used in competitive radioreceptor assays (RRA) after a 3-day culture in 96-well plates with 1-mu-Ci/ml [H-3]glycine. Bovine serum albumin (BSA), modified extensively by incubation with glucose-6-phosphate in vitro to form AGE-BSA, was labeled with I-125 and was used as a model ligand at a concn of 10-mu-g/ml. One unit of AGE was defined as the amount of test protein required to inhibit 50% of the specific binding of [I-125]-labeled AGE-BSA to the AGE-receptors of intact RAW 264.7 cells. Nonlabeled AGE-BSA was used as a specific competitor to construct standard curves. The reproducibility of the assay was assessed at AGE levels equivalent to mean, maximum, and minimum levels of sensitivity for assays run on a single day and over an extended period, and the RRA had a reproducibility (coefficient of variation) between 5.9 and 14.7%. Protease hydrolysis of in vitro glycosylated proteins before assay increases the competitive ability of these proteins in proportion to their glycosylation. Little or no AGE cross-reactivity was detected in native BSA, Amadori-BSA, maleylated BSA, formaldehyde-treated BSA, palmitic acid-BSA, and acetylated low-density lipoproteins (acetyl-LDL). Polyanions such as heparin or fucoidan strongly interfere with this receptor binding assay. In addition, with this RRA, we quantitated AGE formed on proteins other than albumin, such as collagen, LDL, and RNase, modified by glucose in vitro. The development of a novel RRA for AGEs with high sensitivity and specificity for AGEs formed in vitro suggests that such an assay may allow definitive correlations between in vivo AGE development and human pathology or therapeutic intervention to be tested.