Endogenous advanced glycation endproducts (AGEs) include chemically crosslinking species (glycotoxins) that contribute to the vascular and renal complications of diabetes mellitus (DM). Renal excretion of the catabolic products of endogenous AGEs is impaired in patients with diabetic or nondiabetic kidney disease (KD). The aim of this study was to examine the oral absorption and renal clearance kinetics of food AGEs in DM with KD and whether circulating diet-derived AGEs contain active glycotoxins. Thirty-eight diabetics (DM) with or without KD and five healthy subjects (NL) received a single meal of egg white (56 g protein), cooked with (AGE-diet) or without fructose (100 g) (CL-diet). Serum and urine samples, collected for 48 hr, were monitored for AGE immunoreactivity by ELISA and for AGE-specific crosslinking reactivity, based on complex formation with 125 I-labeled fibronectin. The AGE-diet, but not the CL-diet, produced distinct elevations in serum AGE levels in direct proportion to amount ingested (r ‫؍‬ 0.8, P < 0.05): the area under the curve for serum (Ϸ10% of ingested AGE) correlated directly with severity of KD; renal excretion of dietary AGE, although normally incomplete (only Ϸ30% of amount absorbed), in DM it correlated inversely with degree of albuminuria, and directly with creatinine clearance (r ‫؍‬ 0.8, P < 0.05), reduced to <5% in DM with renal failure. Post-AGE-meal serum exhibited increased AGE-crosslinking activity (two times above baseline serum AGE, three times above negative control), which was inhibited by aminoguanidine. In conclusion, (i) the renal excretion of orally absorbed AGEs is markedly suppressed in diabetic nephropathy patients, (ii) daily inf lux of dietary AGEs includes glycotoxins that may constitute an added chronic risk for renal-vascular injury in DM, and (iii) dietary restriction of AGE food intake may greatly reduce the burden of AGEs in diabetic patients and possibly improve prognosis.

Nitric oxide (an endothelium-derived relaxing factor) induces smooth muscle relaxation and is an important mediator in the regulation of vascular tone. Advanced glycosylation end products, the glucose-derived moieties that form noncnzymatically and accumulate on long-lived tissue proteins, have been implicated in many of the complications of diabetes and normal aging. We demonstrate that advanced glycosylation products quench nitric oxide activity in vitro and in vivo. Acceleration of the advanced glycosylation process in vivo results in a time-dependent impairment in endothelium-dependent relaxation. Inhibition of advanced glycosylation with aminoguanidine prevents nitric oxide quenching, and ameliorates the vasodilatory impairment. These results implicate advanced glycosylation products as important modulators of nitric oxide activity and endothelium-dependent relaxation. (J. Clin. Invest. 1991. 87:432-438.)

ABSTRACTzheimer disease (AD) is characterized by deposits of an aggregated 42-amino-acid P-amyloid peptide (I3AP) in the brain and cerebrovasculature. After a concentration-dependent lag period during in vitro incubations, soluble preparations of synthetic .8AP slowly form fibrillar aggregates that resemble natural amyloid and are measurable by sedimentation and thioflavin T-based fluorescence. Aggregation of soluble flAP in these in vitro assays is enhanced by addition ofsmall amounts ofpre-aggregated gamyloid "seed" material. We also have prepared these seeds by using a naturally occurring reaction between glucose and protein amino groups resulting in the formation of advanced "glycosylation" end products (AGEs) which chemically crosslink proteins. AGE-modified flAP-nucleation seeds further accelerated aggregation of soluble flAP compared to non-modiflied "seed" material. To better understand the factors which contribute to amyloid formation and stability, we have studied the in vitro aggregation of soluble synthetically prepared PAP monomers, a process which displays nucleation-dependent kinetics, especially at physiological (nanomolar) concentrations of monomer (refs. 9 and 10; this report). Although millimolar concentrations of (3AP exhibit extensive aggregation within minutes, micromolar and lower concentrations of monomer display a concentration-dependent lag period during which little or no measurable aggregate is formed, followed by a "growth" phase of more rapid aggregation (9-11). This lag period can be eliminated by adding trace amounts of preformed aggregate as "seed" material which serves to immediately induce aggregation (9). Seeding apparently eliminates the need for de novo formation of aggregation nuclei, a much slower process than the subsequent growth of nucleated aggregates. Thus, amyloid seeds can be seen to represent critical protein masses with special structural features capable of nucleating the formation of larger insoluble aggregates of amyloid from pools of soluble P3AP. The nature of these structural characteristics is unknown, but the net result of seeding is significant acceleration in the rate of aggregation compared with unseeded incubations of soluble PAP. By extrapolation from this simple in vitro model to the similar nanomolar concentrations of soluble fAP found in cerebrospinal fluid in vivo, it might be expected that the deposition and accumulation of PAP as amyloid plaques would reflect, in part, the amount or availability of seed material able to nucleate aggregation. Some cases of AD might correspondingly arise as a consequence of increased availability of nucleation seeds in the disease-prone brain relative to normal counterparts not destined to suffer AD at a similar chronological age.In the present communication, we explore the possibility that the formation or stability of amyloid structures which seed PAP aggregation may be enhanced by covalent crosslinks that chemically polymerize components of the aggregate. Under physiological conditions, long-lived proteins beco...

To address potential mechanisms for oxida-

No abstract