Immunochemical detection of N2-[1-(1-carboxy)ethyl]guanosine, an advanced glycation end product formed by the reaction of DNA and reducing sugars or L-ascorbic acid in vitro.
In the Maillard reaction, free amino groups of proteins and nucleic acids react with reducing sugars to form advanced glycation end products (AGE). A major product found in reaction mixtures of guanosine and glucose is N2-[1-(1-carboxy)ethyl]guanosine (CEG), which, therefore, can be used as a marker of advanced glycation of DNA. An enzyme-linked immunosorbent assay (ELISA) was developed to detect and to semi-quantitate nonenzymatic glycosylation of DNA. A polyclonal antiserum was raised against CEG linked to keyhole limpet hemocyanin. A protocol for a competitive ELISA was developed, and the antiserum was tested for crossreactivity. Several unmodified nucleotides and N2-modified guanosine derivatives showed no or negligible crossreactivity. Only very similar structures like N2-(carboxymethyl)guanosine and N2-(1-carboxy-3-hydroxypropyl)guanosine, which have been identified as reaction products of glucose or l-ascorbic acid and guanosine, display significant binding activity. The signal can be totally repressed by free CEG, yet protein-bound CEG is a stronger inhibitor. DNA incubated with d-glucose, dihydroxyacetone, l-ascorbic or l-dehydroascorbic acid shows a signal inhibition indicating the formation of CEG in vitro. The competitive ELISA procedure proved to be a sensitive method which can be used to detect glycation of DNA in vivo.
Seidel
W
W
Institut für Pharmazie, Abteilung Lebensmittelchemie, Ludwig-Maximilians-Universität München, Sophienstrasse 10, 80333 Munich, Germany.
Pischetsrieder
M
M
D000596:Amino Acids / Q000737:chemistry; D000915:Antibody Affinity / Q000187:drug effects; D001205:Ascorbic Acid / Q000737:chemistry*; D003429:Cross Reactions; D004247:DNA / Q000737:chemistry*; D004098:Dihydroxyacetone / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005947:Glucose / Q000737:chemistry*; D017127:Glycation End Products, Advanced / Q000032:analysis*; D006031:Glycosylation; D006151:Guanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis; D007106:Immune Sera / Q000276:immunology
D000596:Amino Acids; D017127:Glycation End Products, Advanced; D007106:Immune Sera; C116679:N(2)-(1-(1-carboxy)ethyl)guanosine; D006151:Guanosine; D004247:DNA; D005947:Glucose; D004098:Dihydroxyacetone; D001205:Ascorbic Acid