Yong Ming Li

Standardizing the immunological measurement of advanced glycation endproducts using normal human serum

Advanced glycation endproducts (AGEs) have been linked to many sequelae of diabetes, renal disease and aging. To detect AGE levels in human tissues and blood samples, a competitive enzyme-linked immunosorbent assay (ELISA) has been widely used. As no consensus or standard research method for the quantitation of AGEs currently exists, nor a universally defined AGE unit available, the comparative quantitation of AGEs between research laboratories is problematic and restricts the usefulness of interlaboratory clinical data. By comparing the cross-reactivities of five different anti-AGE antisera with five different in vitro AGE-modified proteins, we found that the immunological recognition of AGEs by competitive ELISA is both AGE-carrier protein- and anti-AGE antibody-dependent. This suggests that in vitro AGE-modified proteins might not be appropriate standards for AGEs that occur naturally in vivo. Based on our observation that serum AGE levels in the normal human population are consistently within a narrow range and several folds lower than in diabetics, we propose a method to standardize AGE units against normal human serum (NHS). In this new method, one AGE unit is defined as the inhibition that results from 1:5 diluted NHS in the competitive AGE-ELISA; thus the AGE value in NHS is 5 units/ml. This NHS method requires a competitive AGE-ELISA with reasonable sensitivity such that 1:5 NHS produces a 25 to 40% inhibition of anti-AGE antibody binding to immobilized AGE-proteins. By using this standardized method we found that the AGE levels in normal human serum (5.0 +/- 2.2 units/ml; mean +/- SD, n = 34) fit a normal distribution (chi 2-test, p < 0.01), and the serum AGE levels in diabetic patients (20.3 +/- 3.8 units/ml, n = 7) are significantly higher than that of the normal population (p < 0.0001). Since AGE units can now be defined against a universally available standard, NHS, the results of quantitative AGE measurements using this method should be comparable between assays and between different laboratories. Taken together, standardizing the AGE-ELISA protocol as described here provides a simple and quantitative method that should facilitate the expanded application of clinical AGE data.

Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages

Thioglycollate medium (TGM) is widely used as a stimulatory agent to induce non-infectious peritoneal inflammation for elicitation of macrophages from mice and rats. It has been known for a long time that aged, autoclaved TGM is more efficient than freshly prepared TGM, however, the mechanism responsible for this enhanced activity of aged TGM remains obscure. The aging of TGM apparently favors the non-enzymatic reactions between proteins and reducing sugars in TGM that may lead to the generation of advanced glycation endproducts (AGEs). We have found that aged TGM contains 40-fold more AGEs than fresh TGM. The formation of AGEs in TGM was completely blocked by co-incubation with an AGE inhibitor, aminoguanidine. Intraperitoneal injection of aged TGM into rats elicited approximately 2-fold more macrophages than fresh TGM. However, the addition of AGE-modified proteins to fresh TGM increased the yield of peritoneal cells to a level which was significantly higher than both fresh and aged TGM. The injection of AGE-modified proteins alone did not elicit significantly more macrophages than the level of resident peritoneal cells. These results suggest that the formation of AGEs during aging of TGM is responsible for the enhanced macrophage-eliciting activity in aged TGM. AGEs may act as an enhancing agent to augment the existing inflammatory responses. AGE-supplemented TGM may provide an efficient method for eliciting peritoneal macrophages or establishing an inflammatory animal model.