Identification of Amadori-Modified Plasma Proteins in Type 2 Diabetes and the Effect of Short-Term Intensive Insulin Treatment ABDUL JALEEL, PHD 1 PANAGIOTIS HALVATSIOTIS, MD 1 BRIAN WILLIAMSON, PHD 2 PETER JUHASZ, PHD 2 STEPHEN MARTIN, PHD 2 K. SREEKUMARAN NAIR, MD, PHD 1 OBJECTIVE — Growing evidence supports that nonenzymatic glycation products may cause hyperglycemia-induced diabetes complications. Amadori-modified proteins are the intermedi- ate products of nonenzymatic glycation and constitute the forms of glycated proteins in diabetes. The objective of the current study was to utilize two-dimensional gel electrophoresis, Western blot, and mass spectrometry to identify Amadori-modified plasma proteins in type 2 diabetic patients with poor glycemic control and assess the impact of short-term insulin treatment on the glycation of these proteins. RESEARCH DESIGN AND METHODS — We compared eight type 2 diabetic subjects (aged 56  3 years and BMI 29.7  0.9 kg/m 2 ) with an average diabetes duration of 8.5 years (range 3–19) with equal numbers of weight-matched (aged 56  2 years and BMI 30.1  10.0 kg/m 2 ) and lean (aged 58  2 years and BMI 25  00.5 kg/m 2 ) nondiabetic subjects who have no first-degree relatives with diabetes. Two separate blood samples were collected from the type 2 diabetic subjects, one following 2 weeks of withdrawal of all antidiabetic medications (T 2 D-; plasma glucose 12.6  1.0 mmol/l) and another following 10 days of intensive insulin treatment (T 2 D+; plasma glucose 5.5  0.2 mmol/l). Plasma proteins were separated using single and two-dimensional gel electrophoresis. Western blot analysis was performed, and several proteins, which reacted with the Amadori-antibody (1-deoxyfructosyl lysine), were identified by tandem mass spectrometry. RESULTS — No significant differences in the glycation of proteins between the obese and lean groups were noted, but type 2 diabetic patients had several proteins with higher glycation than the control groups. We identified 12 plasma proteins with reduced reaction to the anti-Amadori antibody upon intensive insulin treatment. A significant (P  0.03) difference in Amadori modification was observed between the T 2 D- and control subjects for all these proteins except the Ig light chain. Insulin treatment reduced Amadori modification of albumin (23.2%, P  0.02), fibrin (34.6%, P  0.001), Ig heavy chain constant region (20.7%, P  0.05), transferrin (25.4%, P  0.04), and Ig light chain (13%, P  0.02). In addition, Western blot analysis of two-dimensional gel electrophoresis identified -fibrinogen precursor, -fibrinogen precursor, fibrinogen -B chain precursor, he- mopexin, vitamin D binding protein, and serine protease inhibitor as proteins with a reduced reaction to anti-Amadori antibody upon intensive insulin treatment. CONCLUSIONS — The current approach offers the opportunity to identify Amadori mod- ification of many proteins that may cause functional alterations and offers the potential for monitoring short-term glycemic control in diabetic patients. Diabetes Care 28:645– 652, 2005 H yperglycemia-induced advanced glycation end products (AGEs) are implicated as one of the main un- derlying mechanisms of chronic compli- cations in diabetes (1–9). Nonenzymatic glycation reactions between extracellular proteins and glucose are one of the lead- ing pathways for the formation of AGEs. This involves the condensation reaction of the carbonyl group of sugar aldehydes with the free amino groups or NH 2 termi- nus of proteins, resulting in the formation of a Schiff base. This condensation prod- uct undergoes rearrangement through reversible acid-based catalysis to interme- diate Amadori adducts. AGEs are the complex end products of the irreversible chemical reactions of the Amadori adduct. Recent reviews (8,10,11), based on several published results, support the re- lationship of AGEs with complications of diabetes, and they are specifically impli- cated in retinopathy (12,13), nephropa- thy (14–17), neuropathy (18,19), immunodeficiency (20), and generalized vasculopathy (21,22) in diabetes. AGE formation may modify the functional group of proteins, thus producing abnor- mal interactions between molecules, re- sulting in their altered functions. Studies involving humans (23,24), animals (25,26), and cell culture (27,28) demon- strate a strong relationship between Ama- dori-albumin and diabetes-specific complications. Several markers have been identified for the screening, diagnosis, and monitoring of the disease. The mea- surement of HbA 1c in diabetic patients re- flects glycemic status over the previous 4 – 6 weeks and is routinely used as an index of long-term glycemic control. He- moglobin has a relatively long life in blood, with the average life span of red cells being 120 days. The decay of HbA 1c is also slow, with an average half-life of 29 days (29). In addition to HbA 1c , quantifi- cation of various AGEs are suggested as a long-term assessment of hyperglycemia mainly because of the property of AGEs to ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● From the 1 Endocrinology Research Unit, Mayo Clinic, Rochester, Minnesota; and 2 Applied Bio Systems, Boston, Massachusetts. Address correspondence and reprint requests to K. Sreekumaran Nair, MD, PhD, Mayo Clinic, 200 First St., SW, 5-194 Joseph, Rochester, MN 55905. E-mail: nair.sree@mayo.edu. Received for publication 24 June 2004 and accepted in revised form 9 December 2004. Abbreviations: AGE, advanced glycation end product. A table elsewhere in this issue shows conventional and Syste `me International (SI) units and conversion factors for many substances. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Pathophysiology/Complications O R I G I N A L A R T I C L E DIABETES CARE, VOLUME 28, NUMBER 3, MARCH 2005 645