Anal. Chem. 2010, 82, 3745–3750
Measurement of Urinary Total Desmosine and Isodesmosine Using Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry Osama Albarbarawi,† Alun Barton,† ZhaoSheng Lin,‡ Eddie Takahashi,‡ Ajay Buddharaju,† Jeffrey Brady,†,‡ Douglas Miller,‡ Colin N. A. Palmer,§ and Jeffrey T.-J. Huang*,†,‡,§ Translational Medicine Research Collaboration, TMRC Laboratory, James Arrott Drive, Dundee, DD1 9SY, Pfizer Inc., United Kingdom, and Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee The current LC-MS based desmosine/isodesmosine (DES/ IDS) assays may be unsatisfactory for clinical use due to lack of an appropriate internal standard or low throughput. A fast and reliable LC-MS method using a D5-DES as an internal standard for measuring urinary total DES/IDS was developed and validated in this study. The reportable range of this assay was 1.0 and 480.0 ng/mL. The intra- and interassay imprecision, accuracy, and recovery for quality control samples were within acceptable range (8 dilution ratio is not recommended. These results suggest that the reportable concentrations for urinary total DES/IDS may go up to approximately 480 ng/mL of DES/IDS following an 8-fold dilution when necessary. Analytical Performance: Precision and Accuracy/Recovery. The intra- and interassay precision and accuracy/recovery of three validation samples (low, mid, and high QC samples) were determined in triplicate per day, during three different days. The intra-assay imprecision (%CV) and accuracy (%bias) for total DES/ IDS ranged from 5.0 to 16.4 and -11.1 to 0.3, respectively (SI Table S-3). The interassay imprecision and accuracy percentages ranged from 1.6 to 5.1 and from -1.1 to -0.2, respectively. The total recovery of the assay was also analyzed through the analysis of mid- and high-validation samples. Intra- and interassay recovery for total DES/IDS ranged from 88.9 to 99.0% and 98.9 to 99.8%, respectively. SPE recovery was also assessed using urine samples spiked with mid and high levels of DES prior to or after SPE. The percentage of total DES/IDS recovery ranged from 109.6 to 111.6% (SI Table S-4). Stability. Short-term stability (up to 20 h at RT and at 4 °C), as well as stability following 3 F/T cycles (using freshly collected urine stored at 4 °C for no more than 4 h) was assessed using three urine samples (run in triplicate) from apparently healthy
Figure 3. Urinary total DES/IDS as a biomarker for COPD (A) The level of urinary total DES/IDS in COPD patients were about 3 times higher than those in healthy volunteers (p ) 1.2 × 10-5; t test). The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. (B) Except one patient with COPD, the urinary total DES/ IDS levels were well correlated to the urinary free DES/IDS levels (R2 ) 0.85, Pearson’s correlation).
donors. No significant reduction of DES/IDS was observed following storage at room temperature (20 h, 98.9% recovery), or 4 °C (20 h, 100.7% recovery) or after three freeze/thaw cycles (106.5% recovery) (SI Table S-4). Creatinine Normalized Urinary Total DES/IDS Levels in Healthy Individuals and COPD Patients. To assess the utility of this method for measuring total urinary DES/IDS as a biomarker for COPD, we analyzed a total of 36 urine samples (random urine samples) including 18 COPD patients and 18 age and gender matched apparently healthy donors (Table 2, first cohort). The levels of urinary total DES/IDS in all healthy volunteers and COPD patients were above the LLOQ ranging between 1.1 and 49.4 ng/mL. The level of total DES/IDS was normalized to the levels of urinary creatinine. For healthy volunteers, the level of urinary total DES/IDS were 6.8 ± 3.2 ng/ mg creatinine (mean ± SD). Urinary total DES/IDS in COPD Analytical Chemistry, Vol. 82, No. 9, May 1, 2010
3749
patents were about 3 times higher than those in healthy volunteers (COPD: 20.4 ± 14.9 ng/mg creatinine; p ) 1.2 × 10-5; t test) (Figure 3A). Despite a small number of healthy smokers in the study, it was noticed that four healthy smokers had higher urinary total DES levels (10.8 ± 3.1 ng/mg creatinine) creatinine compared to nonsmokers and ex-smokers among the healthy group (5.6 ± 2.1 ng/mg creatinine). The smoking related elevation of urinary total DES was in line with previous reports.7,8 In comparison to the results from other studies using LC-MS-based methods, the results for healthy volunteers reported in this study was lower compared to the results from Boutin and colleagues (13.2 ± 1.9 ng/mg creatinine for healthy smokers and 14.9 ± 2.9 ng/mg for healthy nonsmokers22), in which a stable isotopic desmosine standard was also included. In contrast, we also found that the levels of urinary total DES/IDS in COPD patients were twice as high as those reported in the same study.22 The reason for the discrepancy is not clear, but it is likely due to the small size of cohorts, the nature of spot urine samples used, methods used to measure creatinine, and/or different disease severity of patients recruited in the studies. A larger cohort with whole day urine samples (or multiple visits if spot urine samples are preferred) is urgently required to establish the validity of this marker for disease progression and pharmacodynamic monitoring for compounds inhibiting proteases that cause lung degradation. We recognized that one potential limitation of applying this method in clinical chemistry laboratories is the requirement of acid hydrolysis, where it requires 24 h for sample preparation and the use of 12 M hydrochloric acid adds another degree of health and safety requirements. As free DES and IDS has been shown to be detectable in urine samples,6 we also investigated if free DES/IDS levels can be used to estimate elastin degradation. To do so, a second cohort consisting of a total of 40 samples (20 COPD patients and 20 healthy volunteers, see Table 2, second cohort, for the demographics) were processed and analyzed in the same method procedure except that the acid hydrolysis step was omitted. Two free desmosine levels were below LLOQ and were excluded. We found that except one patient with COPD, the urinary total DES/IDS levels were well correlated to the urinary free DES/IDS levels (R2 ) 0.85, Pearson’s correlation, Figure
3750
Analytical Chemistry, Vol. 82, No. 9, May 1, 2010
3B). However, the differences between the two groups in terms of free DES/IDS were not as significant as the total DES/IDS (free DES/IDS: p ) 0.001 vs total DES/IDS: p ) 1.2 × 10-5). There was a substantial overlap between the two groups in urinary free DES/IDS levels. These results suggests that despite urinary free and total DES/IDS levels being highly correlated, it is preferred to measure urinary total DES/IDS level as a biomarker for elastin degradation for COPD. Although the scope of the current study focuses on urinary DES/IDS, it is perceivable that total DES/IDS measurements in other body fluids such as sputum (to a lesser degree, plasma) may reflect more directly to the lung pathologies in respiratory diseases. Some initial studies by Turino and associates have shown some promising results.5,6 Further effort for analytical validation of these methodologies and clinical validation of sputum/plasma total DES/IDS as a biomarker for respiratory diseases are urgently required. CONCLUSION In summary, we have developed and validated a fast and reliable LC-MS method using stable isotopic DES as an internal standard for measuring total urinary total DES/IDS. Our results demonstrate that the LC-MS/MS method provides sensitive, reproducible, and accurate quantification of total DES/IDS in human urine samples as a biomarker for monitoring elastin degradation in diseases such as COPD. ACKNOWLEDGMENT We thank Professor Simon Robins from University of Aberdeen and Dr. Ian Kay from Thermo Scientific for their valuable suggestions and technical support. The first two authors contributed equally to this work. SUPPORTING INFORMATION AVAILABLE Figures S-1-S-4, Tables S-1-S-5. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 19, 2010. Accepted March 4, 2010. AC100152F
