Authorship:
J.M.W. van den OUWELAND1, M. SPANBROEK2, R. JANSSEN2, A. BEIJERS1, H. van DAAL1
Department of Clinical Chemistry1 and Pulmonary Disease2, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands
| Short Abstract Desmosine is a promising biomarker for estimating activity of elastin degradation in patients with COPD, although its clinical validity remains uncertain as reliable and sensitive assays are lacking. An LC-MS/MS method for measuring plasma desmosine was developed and validated. Sample preparation consisted of acid hydrolysis, cellulose SPE using D4-DES as IS, followed by C18 chromatography and MS-analysis using SRM. Measuring range was 0.1-10 ng/ml, imprecision <10%, with recoveries within 80-120%. Plasma desmosine levels from COPD patients were significantly higher compared with healthy individuals. The LC-MS/MS assay appears a valuable tool to assess the potential of desmosine as a biomarker for monitoring disease activity in COPD and to study the effect of therapeutic interventions. |
Long Abstract
Introduction: Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. Its degradation gives rise to desmosine (DES) and isodesmosine (IDS) in body fluids. DES and IDS are promising biomarkers for estimating activity of elastin degradation, although their clinical validity and utility remain uncertain due to the lack of reliable and sensitive assays. The objective of this study was to develop and validate a stable isotope dilution LC-MS/MS method for measurement of DES and IDS in urine and plasma.
Methods: The method is based on spiking with a deuterium labeled desmosine standard (d4-DES) to urine or plasma sample, acid hydrolysis (o/n at 110 °C), SPE using cellulose, C18 chromatography using 5mM NH4COOH in H2O/MeOH+ 0,1% HFBA and positive ESI MS/MS analysis (Xevo TQS, Waters) by SRM monitoring of transition ions 526-481 for DES, 526-397 for IDS and 530-486 for d4-DES. Lower limit of quantitation (LLOQ), imprecision profile, linearity and recovery were established. Plasma total DES levels were determined in healthy non-smokers (n=30), (passive) smokers (n=45), and patients with COPD (n=72).
Results: DES and IDS were base-line separated, eluting at Rt 6.6 and 6.3 min, respectively. LLOQ was 0.1 ng/mL. Intra- and inter-assay imprecision were <10%. The assay was linear up to 10 ng/mL. Recoveries were 96-118% for IDS and 100-118% for DES. Post-column injection of d4-DES IS revealed no significant ion-suppression for either metabolite. Plasma desmosine levels from smokers and patients with COPD were significantly higher compared with healthy individuals (p<0.0015 and p<0.0001, respectively).
Conclusion: We have developed a sensitive and specific assay for the measurement of DES and IDS in human urine and plasma. This method can be used to assess the potential of DES and IDS as biomarkers for estimating disease activity in COPD and to study the effect of therapeutic interventions.