Circulating markers of protein glycation, oxidation and nitration in type 2 diabetes and effect of decline in renal function

George Jerums, Sianna Panagiotopoulos,Richard J. MacIsaac,Dennis K. Yue, Greg R. Fulcher, Matthew Roberts, James R Larkin, Antonysunil Adaikalakoteswari, Naila Rabbani and Paul J. Thornalley

Poster at American Diabetes Association 71st Scientific Sessions, San Diego, California, USA (2011)

Diabetes 60 Supp 1 A166. DOI: 10.2337/db11-478-715


Impairment of renal function in diabetes has been associated with raised serum levels of advanced glycation endproducts (AGEs) and, conversely, hyperfiltration has been associated with low circulating levels of AGEs. Proteins are modified by glycation, oxidation and nitration in vivo. Serum contains glycation, oxidation and nitration adducts in protein and related free adducts (glycated, oxidized and nitrated amino acids) – the latter formed by proteolysis of damaged proteins. The aim of this study was to explore the relationship of circulating markers of protein damage to glomerular filtration rate (GFR) in type 2 diabetes.

Study participants had type 2 diabetes and 5 categories of estimated GFR: 15-30, 31-60, 61-90, 91-120 and > 120 ml/min/1.73 m2 (n = 10-15 per category). Fasting serum samples were obtained prior to a routine clinic visit and stored at -20°C until assay. Fourteen glycation, oxidation and nitration adducts were quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry - free adducts by analysis of a 12 kD ultrafiltrate and protein adducts after exhaustive enzymatic hydrolysis of serum protein. Study group means are compared.

Overall, free adducts were more sensitive to decline in GFR than protein adducts. For AGE free adducts, methylglyoxal-derived hydroimidazolone (MG-H1) increased from 234 to 2611 nmol/L, Nε-carboxymethyl-lysine (CML) from 60 to 277 nmol/L and pentosidine from 0.15 to 2.04 nmol/L with decline of GFR (P<0.001). For oxidation adducts, dityrosine increased from 0.89 to 6.36 nmol/L and N-formylkynurenine (NFK) from 5.4 to 12.2 nmol/L whereas methionine sulfoxide (MetSO) decreased from 6.2 to 3.4 µmol/L. The nitration free adduct, 3-nitrotyrosine (3-NT), increased from 4.5 to 21.8 nmol/L. In serum protein, AGEs showed modest increases, dityrosine, NFK and 3-NT were unchanged whereas MetSO increased with decline in GFR.

These results are consistent with modest increases in glycation and methionine oxidation of protein in the circulation and marked decreases in clearance of most glycation, oxidation and nitration free adducts with decline in GFR in type 2 diabetes.