Febs Letters Volume 129, Number 1-2./Volume 130, Number 1-2. [antikvár]

Volume 83, number 1 FEBS LETTERS November 1981 NON-ENZYMATIC GLYCOSYLATION OF HUMAN SERUM LIPOPROTEINS Elevated e-Iysine glycosylated low density lipoprotein in diabetic patients E. SCHLEICHER, T. DEUFEL and 0. H. WIELAND Klmisch-Chemisches Institut und Forschergruppe Diabetes, Aliademisches Lehrkranlcenhaus München-Schwabing, 8000 München 40, Kölner Platz 1, FRG Received 14 April 1981 1. Introduction Tlie 'minor hemoglobins' (HbAjj_(,) were the first examples to show that glucose can react non-enzy-matically with proteins in the human body to form stable glycosyl—protein adducts. Other proteins have been observed to undergo non-enzymatic glycosylation, such as the eye lens crystallins [1], red cell membrane proteins [2,3], serum albumin [4-6]. The amount of HbAj3_(, is increased in diabetic patients depending on the level of hyperglycemia; this holds also for glycosylated albumin [7,8], and erythrocyte membrane proteins [2,3]. Apart from the diagnostic value of HbAi and glycosyl—albumin as tools for long-term control of diabetes, protein glycosylation has attracted special interest with regard to the role it may play in the development of the late complications of diabetes. In these studies we have found that glucose is covalently bound to e-amino groups of lysine of human apo-lipoproteins upon incubation, in vitro. Moreover, we could show that the level of glycosylated apoprotein B (apo-B) of the low density lipoproteins (LDL) is increased in the serum from diabetic patients. 2. Materials and methods Human serum albumin and reagents for the quantitative determination of apo-A and apo-B by immunodiffusion were from Behringwerke (Marburg). Immobilised heparin (lipotype) was purchased from Dedicated to Professor Helmuth Hoizer on the occasion of his 60th birthday Panchem (Kleinwallstadt). Chemicals used for polyacrylamide gel electrophoresis were from Serva (Heidelberg), all other chemicals were from Merck (Darmstadt). HPLC analysis was done on a Waters apparatus (Milford USA). D-iU-'^C]Glucose was from Radiochemical Centre (Braunschweig). To remove any radioactive contaminants which are prone to react rapidly with albumin (unpublished) and could invalidate the in vitro labelling experiments [9], the preparation was pre-purified by dissolving 6.9MmolD-[U-"'C]glucosein 1 ml phosphate-buffered saline (PBS) contaming 2 mg human serum albumin. After 36 h incubation at 37°C the albumin was precipitated by acetone, the supernatant evaporated, and the glucose purified by gel-filtration on Sephadex G-25. 2.1. Purification of lipoproteins Human serum lipoproteins for incubation studies were purified by ultracentrifugation essentially accordmg to [ 10]. Apo-A and B content was estimated by radial immunodiffusion. LDL from normal and diabetic patients were isolated as follows: 0.5 ml serum was applied to a heparin column (Panchem) and the (3-lipoproteins were eluted according to tKe supplier's instructions. After adjusting the density to 1.065 by extensive dialysis the (3-lipoproteins were flotated by ultracentrifugation at 15°C for 18 h at 150 000 X g. LDL and VLDL were separated under the same conditions at a density of 1.006. LDL was precipitated and delipidated according to [11]. LDL from patients was also purified by immuno-adsorption [12] on a column containing Sepharose-bound anti-apoprotein B immunoglobulin. The immobilized anti-apoprotein B was kindly provided Elsevier/North-Holland Biomedical Press 1