ENZYMATIC FLUOROMETRIC ASSAY FOR EPSILON(GAMMA-GLUTAMYL)LYSINE ISODIPEPTIDES - A NEW ASSAY FOR PROTEOLYTIC FRAGMENTS OF PROTEINS CROSS-LINKED BY FACTOR-XIII AND OTHER TRANSGLUTAMINASES

A fluorescent method for the quantitation of epsilon(gamma-glutamyl)ly sine isodipeptides is described which is applicable to the quantitatio n of the free isodipeptides in human plasma and in proteolytic digest of human platelet lysates. The possible diagnostic importance of free epsilon(gamma-glutamyl)lysine isodipeptides in plasma revealed the nec essity of developing a relatively simple method suitable for the fast screening of large numbers of samples. Samples are deproteinised by Ce ntrifree micro partition system, then purified by cation-exchange chro matography and lyophilised. Aliquots of the reconstituted samples are incubated with gamma-glutamylamine cyclo-transferase, the enzyme which specifically catalyses the conversion of epsilon(gamma-glutamyl)lysin e to free lysine and 5-oxo-L-proline. The L-lysine is decarboxylated a nd the cadaverin formed is extracted by pentane-1-ol. The primary amin e produces a stable product with fluorescamine which is measured fluor imetrically. The epsilon(gamma-glutamyl)lysine isodipeptide standard c urves were linear in a concentration range of 0-100 mu mol/l. Imprecis ion of measurement was 34% at 1.4 mu mol/l and 18% at 24.6 mu mol/l co ncentrations. Results correlated with the HPLC technique described pre viously (r=0.798). Median (and ranges) of the isodipeptide levels in p lasma were 2.4 (0-9.2) mu mol/l (n=14) in apparently healthy individua ls, 11.8 (0-80.7) mu mol/l (n=23) in patients with deep vein thrombosi s and 0.9 (0.1-7.9) mu mol/l (n=6) in patients with acute myocardial i nfarction.