The detection and quantitation of free desmosine and isodesmosine in human urine and their peptide-bound forms in sputum

Desmosine (D) and isodesmosine (I), the intramolecular crosslinking amino acids that occur in chains of elastin, have now been found in free form in human urine. Until now, these amino acids (M_r = 526) were found to occur in urine only as higher molecular weight (M_r = 1,000-1,500) peptides. Thus, the previously used analytical methods required, as the first step, acid hydrolysis of the urine at elevated temperature to liberate D and I from their peptides. The analytical method described here uses HPLC followed by electrospray ionization MS for the detection and quantitation of free D and I in unhydrolyzed urine. Identities of both D and I were established by their retention times on LC and by their mass ion at 526 atomic mass units, characteristic of each compound. The sensitivity of the method is 0.10 ng. The average values of free D and I in the urine of seven healthy subjects were 1.42 ± 1.16 and 1.39 ± 1.04 μg/g of creatinine, respectively. After acid hydrolysis of the urine, the amounts of D and I were 8.67 ± 3.75 and 6.28 ± 2.87 μg/g of creatinine, respectively. The method was also successfully used to measure peptide-bound D and I levels in the sputum of patients with chronic obstructive pulmonary disease.