Oxford Research Unit, Open University, Boars Hill, Oxford. OX1 5HR. Advanced glycation end-products (AGEs) include a variety of compounds formed from the ...
Biochemical Society Transactions ( 1 993) 21
93s
Fig. 2. Conversion of AP to AGE requires free metal ions.
NESSAR AHMED, JASON LIGGINS and ANNA J. FURTH Oxford Research Unit, Open University, Boars Hill, Oxford. OX1 5HR. Advanced glycation end-products (AGEs) include a variety of compounds formed from the further reaction of the .," . Amadori product (AP) of protein and sugar. Glycation is the non0 2 4 6 8 1 0 1 2 enzymatic reaction of a reducing sugar carbonyl with the a or EDays o f i n c u b a t i o n amino group of a protein, to form a labile Schiff base which then undergoes an Amadori rearrangement to form Amadori product Glucated BSA was dialysed free of sugar and then re-incubated at [l]. The rate of AP formation is dependent on the percentage of pH7.4 in the absence of sugar in lOmM HEPES buffer (i.e. O.OM reducing sugar present in open chain form, since a free carbonyl phosphate) or phosphate buffer of increasing concentration. group is needed for glycation. That this is true also for AGE ________________________________________-------------------formation can be demonstrated by the in vitro glycation of BSA, This was confirmed by exhaustive dialysis of glucated bovine serum albumin (lOmg/ml), by 0.5M glucose or fructose at BSA (4 days at 4OC with 8 changes of dialysis buffer, each at 37OC and pH 7.4. Figure 1 shows the rate of formation of least 4 hours apart) followed by re-incubation at 37OC in sugarfluorescent AGEs, as detected at the wavelengths optimum for free phosphate buffer of increasing concentration. Fluorescent pentosidine, which is the major fluorophore of in vitro glycated AGES were produced in direct proportion to the phosphate buffer lysozyme [2]. From the different scales in Figs la and b, it is concentration and thus to the concentration of free metal ions in clear that fructose (0.7% acyclic in solution) forms fluorescent the incubation medium (Fig 2). It therefore appears that when no AGE's faster than glucose (0.002% acyclic [3]). free metal is available no further AGEs are formed. Thus the Figure 1 also shows that a component of phosphate buffer effect of MCO on AGE formation in BSA is at the post-Amadori catalyses AGE formation, since fluorescence increases with stage. This supports the suggestion by Baynes, that it is this later buffer concentration in a dose-dependent manner. A similar part of the 'glycation sequence' that is the fixative, the oxidation effect was observed in the formation of crosslinked AGEs by step making the overall reaction irreversible under physiological lysozyme. Doubling the phosphate buffer concentration from conditions [5]. 0.1M to 0.2M at pH7.4 approximately doubled the percentage of Metal-catalysed oxidation has also been implicated in the lysine converted to oligomer, i.e. from 1.3% to 3.4% with formation of crosslinks, pentosidine and carboxymethyllysine in glucose and from 14% to 24% with fructose. Apparent activation collagen [4,6], and of glucose-inducedfluorescence in BSA (325by phosphate buffer has also been reported for the formation of 395nm excitation, 430-470nm emission) [7]. With BSA, crosslinks, fluorophores and carboxymethyllysine in glycated fluorescence increased even after 24 hours prior dialysis to collagen [4]. remove free sugar, and experiments with superoxide dismutase, Sodium phosphate buffer (0.1M at pH7.4) contains traces catalase and chelators suggested that the active principle in MCO of free metal ions, to a calculated maximum of 4.7pM copper and is the hydroxyl radical [7]. A similar role for the hydroxyl radical 5.7pM iron. To see whether metal-catalysed oxidation (MCO) has been suggested in the glucose-induced fragmentation of BSA was responsible for the apparent activation by phosphate, we [8,91. repeated the in vitro glycation of BSA in the presence of the In conclusion, our work c o n f i i s that metal-catalysed metal chelator DTPA. Alternatively we replaced the phosphate oxidation is involved in AGE formation in BSA and lysozyme buffer with HEPES, which has free metal contaminants in the and that with BSA its effect is primarily post-Amadon. nanomolar region. In both cases we found production of AP but no AGEs, suggesting that here, MCO is primarily a postWe are very grateful to Professor Baynes for sight of Amadori event. manuscripts before publication, and to the Glenn Foundation for ________________________________________--------------------Medical Research for support to J.Liggins. Fig 1. Increasing phosphate buffer concentration promotes formation of AGE's. Monnier, V.M. (1989) (la) in The Maillard Reaction in Ageing, Diabetes and Nutrition, 1
+0.3M +0.2M +0.1M
8 150
+O.OSM
100
P 5
m, m.
Y
p 0)
6
z 0
5
10 15 20
(ed. Baynes J.W. & Monnier, V.M.) Alan R Liss, New York, ppl-22. Dyer, D.G., Blackledge, J.A.,Thorpe, S.R. & Baynes, J.W. (1991) J. Biol. Chem.%, 11654-60. Bum, H.F. & Higgins, P.J. (1981) Science 222-224. Fu, M.X., Knecht. K.J., Thorpe, S.R. & Baynes, J.W., (1992) Diabetes 42-48. Baynes, J.W. (1991) Diabetes, 4Q.405-412. Chace, K.V., Carubelli, R. & Nordquist, R.E., (1991) Arch. biochem. Biophys. 28&121,473-480. Le Guen, C.A., Jones, A.F., Bamett. A.H. & Lunec, J. (1992) Ann. Clin. Biochem. B. 184-189. Hunt, J.V.,Dean,R.T. & Wo1ff.S.P. (1988) Biochem. J. 205-212. Kawakishi, S., Okawa, Y. & Uchida K. J. Agric. Food Chem. 3, 13-17.
0
5 10 15 2 0
days o f
days o f
incubalion
incubation
BSA (lOmg/ml) was incubated with 0.5M sugar in phosphate buffer, pH7.4, of increasing concentration.
m,
