Single-column high-performance liquid chromatographic-fluorescence detection of immature, mature, and senescent cross-links of collagen.
A high-performance liquid chromatographic-fluorescence detection method of reducible (immature) and nonreducible (mature and senescent) cross-links of collagen was established without the use of a radioisotope and preliminary fractionation step. This method used a gradient elution procedure of sodium citrate buffer containing 7% ethanol. The reducible cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible cross-link (histidinohydroxylysinonorleucine) were detected by O-phthalaldehyde derivatization with the postcolumn method, whereas other nonreducible cross-links (pyridinoline, deoxypyridinoline, and pentosidine) were detected by natural fluorescence. The linear ranges of contents of the O-phthalaldehyde derivative cross-links and the natural fluorescent nonreducible cross-links were 20-600, 5-500 (pyridinoline, deoxypyridinoline), and 0.2-20 pmol (pentosidine), respectively. Tissue containing 1-2 mg dry wt of collagen was adequate for duplicate analyses of the reducible and nonreducible cross-links. An equivalent of 0.25 mg of hydrolyzed collagen could be analyzed by this HPLC system. Using this system, age-related changes in the cross-links of collagen from human connective tissues were also investigated.
Saito
M
M
Institute of DNA Medicine, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Tokyo, Minato-ku, 105, Japan.
Marumo
K
K
Fujii
K
K
Ishioka
N
N
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging; D000596:Amino Acids / Q000032:analysis; D001120:Arginine / Q000031:analogs & derivatives / Q000032:analysis; D002358:Cartilage, Articular / Q000737:chemistry / Q000254:growth & development*; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003094:Collagen / Q000737:chemistry* / Q000302:isolation & purification; D003432:Cross-Linking Reagents; D018483:Diaphyses / Q000737:chemistry / Q000254:growth & development*; D004151:Dipeptides / Q000032:analysis; D005269:Femur / Q000737:chemistry / Q000254:growth & development; D006639:Histidine / Q000031:analogs & derivatives / Q000032:analysis; D006801:Humans; D007202:Indicators and Reagents; D007223:Infant; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity; D012867:Skin / Q000737:chemistry / Q000254:growth & development*; D013050:Spectrometry, Fluorescence / Q000379:methods
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004151:Dipeptides; D007202:Indicators and Reagents; C075793:histidinohydroxylysinonorleucine; C007428:5,5'-dihydroxylysylnorleucine; C010991:delta-hydroxylysylnorleucine; D006639:Histidine; C015484:pyridinoline; C036020:deoxypyridinoline; D003094:Collagen; D001120:Arginine; C062187:pentosidine; D008239:Lysine