engleski

Investigation into the molecular nature of human skin collagen fluorescence that is implicated as a prognostic marker of cardiovascular disease in diabetes and uremia

Protein-linked long-wavelength fluorescence at 440 nm upon excitation at 370 nm (LW) has commonly been used as marker for the Maillard reaction in vivo. Recent evidence suggests that LW fluorescence of human skin selectively predicts coronary artery calcium deposition and mortality in patients with diabetes and renal disease. Here we initiated studies into the structural elucidation of fluorophore (LW1) identified as a single major peak in HPLC chromatograms from enzymatic digests of human skin collagen. LW1 increased with age and was strongly catalyzed by diabetes and end-stage renal disease. However, LW1 did not co-chromatograph with other identified fluorophores including standards of the cross-links, pentosidine, K2P, and a HNE-derived pyrrole, as well as fluorophoric adducts, argpyrimidine and ST29. The acid-labile LW1 was purified by HPLC from enzymatic digests of ≈ 75 g human skin obtained at autopsy from patients with diabetes and end-stage renal disease. Partialy characterization of LW1 showed a UV maximum at 348 nm and molecular masses at m/z= 312, 447, 624 by ESI-LC/MS. 1H-NMR showed three aromatic protons at 8.6 (singlet), 8.0 (doublet) and 6.9 (doublet) ppm of which the later two protos were found coupled by TOCSY. A 13C-1H NMR correlation experiment (HETCOR) showed the three protons were coupled to 13C signals at 110, 138, and 95 ppm, respectively. In the aliphatic region, protons identified at 2, 6 ppm coupled to a carbon at 14 ppm suggested a methyl group attached to an aromatic ring. Multiple 1H signals at 3.5 and 4.8 ppm and 13C between 45 and 65 ppm suggest the presence of the epsilon protons in lysine. In further agreement, these signals correlated with three groups of 1H signals between 1.5 to 2.3 ppm consistent with the presence of lysine. 1H signals btween 3.6 to 3.8 ppm coupled to 13C signals between 55 to 80 ppm were in agreement with values characteristic of sugar molecule. Overall, the results are consistent with a pyridinium-type of molecule, but the final structure is still elusive. Because LW fluorescence can be monitored non-invasively in skin, this research may have broad implications for the development of novel methodologies for the assessment of risk for macrovascular disease progression in diabetes.

human skin; fluorescence; diabetes; Maillard

nije evidentirano