Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products.
Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.
Hegele
Jörg
J
Nestlé Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. joerg.hegele@rdls.nestle.com
Buetler
Timo
T
Delatour
Thierry
T
D000818:Animals; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D004798:Enzymes / Q000378:metabolism; D006851:Hydrochloric Acid; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008892:Milk / Q000737:chemistry*; D015394:Molecular Structure; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D002241:Carbohydrates; D004798:Enzymes; D011506:Proteins; D008239:Lysine; D006851:Hydrochloric Acid