MEASUREMENT OF URINARY DESMOSINE BY ISOTOPE-DILUTION AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY - CORRELATION BETWEEN ELASTASE-INDUCED AIR-SPACE ENLARGEMENT IN THE HAMSTER AND ELEVATION OF URINARY DESMOSINE

The accuracy of methods employed to measure the elastin-specific crosslinks, desmosine (DES) and isodesmosine (IDES), has been called into question because contaminants In the urine may cause elevated values. In the present study urine samples were spiked with a known amount of [C-14]DES and refluxed in 6 N HCl. Sephadex G-15 chromatography of the hydrolyzed urine was employed to remove contaminants. DES and IDES were quantified by high performance liquid chromatography (HPLC) as well as by amino acid analysis. The amount of isotope recovered was used to determine losses during the overall procedure and the isotope dilution to calculate the amounts of endogenous DES and IDES originally present in the urine. Because similar values were obtained by both methods, the more rapid HPLC method was used for all succeeding analyses. In one experiment, the DES amounts in urine collected from hamsters for 3 days after intratracheal treatment with human neutrophil elastase (300-mu-g) or porcine pancreatic elastase (300-mu-g) were 0.212 +/- 0.012 (mean +/- SEM, two measurements on a single pool) and 0.816 +/- 0.005 (two measurements) mu-g per hamster per day, respectively. Urine from control hamsters had a mean value of 0.074 +/- 0.008 (eight measurements) mu-g per hamster per day. The HNE- and PPE-treated hamsters had mean linear intercept values of 119 and 159% of control values, respectively, giving a positive correlation between increase in airspace size and elevation of urinary DES. For eight normal men 25 to 68 yr of age who were never-smokers, mean +/- SEM 24-h urinary values were 13.3 +/- 2.3-mu-g DES and 11.3 +/- 1.7-mu-g IDES; DES/creatinine and IDES/creatinine ratios were 6.5 +/- 0.7-mu-g and 5.5 +/-0.4-mu-g per g creatinine, respectively. Values for subjects remained constant when measured on urine collected on different days. Hamster and human DES values are as much as 20-fold and 10-fold lower, respectively, than previously reported values. This assay should prove useful in studying conditions when DES excretion may be elevated.