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 [3H]glycine. Bovine serum albumin (BSA), modified extensively by incubation with glucose-6-phosphate in vitro to form AGE-BSA, was labeled with 125I and was used as a model ligand at a concn of 10 micrograms/ml. One unit of AGE was defined as the amount of test protein required to inhibit 50% of the specific binding of [125I]-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Radoff
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Laboratory of Medical Biochemistry, Rockefeller University, New York, New York.
Makita
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Vlassara
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H
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D005998:Glycine / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D006031:Glycosylation; D007457:Iodine Radioisotopes; D007700:Kinetics; D008264:Macrophages; D011869:Radioligand Assay / Q000379:methods; D000067759:Receptor for Advanced Glycation End Products; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011971:Receptors, Immunologic; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014316:Tritium
D006023:Glycoproteins; D007457:Iodine Radioisotopes; D000067759:Receptor for Advanced Glycation End Products; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D014316:Tritium; D012710:Serum Albumin, Bovine; D005998:Glycine