Shuren Ma

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 (Mr = 526) were found to occur in urine only as higher molecular weight (Mr = 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.

Quantitation of desmosine and isodesmosine in urine, plasma, and sputum by LC–MS/MS as biomarkers for elastin degradation

The aim of this study is to develop a standardized LC-MS/MS method for accurate measurement of desmosine (DES) and isodesmosine (IDS) in all body fluids as biomarkers for in vivo degradation of matrix tissue elastin in man and animals. A reproducible three-step analytical procedure: (1) sample hydrolysis in 6N HCl, (2) SPE by a CF1 cartridge with addition of acetylated pyridinoline as internal standard (IS), and (3) LC/MSMS analysis by SRM monitoring of transition ions; DES or IDS (m/z 526-481+397) and IS (m/z 471-128) was developed. The method achieves accurate measurements of DES/IDS in accessible body fluids (i.e. urine, plasma, and sputum). LOQ of DES/IDS in body fluids is 0.1 ng/ml. The % recoveries and reproducibility from urine, plasma, and sputum samples are above 99 ± 8% (n = 3), 94 ± 9% (n = 3) and 87 ± 11% (n = 3), with imprecision 8%, 9% and 10%, respectively. The proposed method was applied to measure DES/IDS in body fluids of patients with chronic obstructive pulmonary disease (COPD) and healthy controls. Total DES/IDS in sputum and plasma is increased over normal controls along with the free DES/IDS in urine in patients. DES/IDS can be used to study the course of COPD and the response to therapy. This practical and reliable LC-MS/MS method is proposed as a standardized method to measure DES and IDS in body fluids. This method can have wide application for investigating diseases which involve elastic tissue degradation.

Matrix elastin: a promising biomarker for chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a major health problem worldwide and is now the third leading cause of death in the United States. There is a lack of therapies that can stop progression of the disease and improve survival. New drug discovery can be aided by the development of biomarkers, which can act as indicators of severity in the course of the disease and responses to therapy. This perspective brings together the laboratory and clinical evidence, which suggest that elastin degradation products can fulfill the need for such a biomarker. Elastin is a recognized target for injury in COPD. The amino acids desmosine and isodesmosine exist only in matrix elastin; can be measured specifically and sensitively in plasma, urine, and sputum; and indicate changes in the systemic balance between elastase activity and elastase inhibition brought on by the systemic inflammatory state. The biomarker levels in sputum reflect the state of elastin degradation in the lung specifically. Clinical data accumulated over several decades indicate correlations of desmosine and isodesmosine levels with COPD of varying severity and responses to therapy.

AEROSOLIZED HYALURONAN LIMITS AIRSPACE ENLARGEMENT IN A MOUSE MODEL OF CIGARETTE SMOKE–INDUCED PULMONARY EMPHYSEMA

This study was designed to determine if aerosolized hyaluronan (HA) could prevent airspace enlargement and elastic fiber injury in a mouse model of cigarette smoke–induced pulmonary emphysema. Compared to untreated/smoked controls, HA-treated animals showed statistically significant reductions in mean linear intercept (54 versus 65 μm; P < .001) and elastic fiber breakdown products (desmosine and isodesmosine) in bronchoalveolar lavage fluid (0.3 versus 7.0 ng/mL; P < .05). As in previous studies, the aerosolized HA showed preferential binding to elastic fibers, suggesting that it may protect them from injury. These findings support further investigation of the potential use of HA as a treatment for pulmonary emphysema.

Alpha-1 Antitrypsin Augmentation Therapy and Biomarkers of Elastin Degradation

Abstract Background: Intravenous alpha-1 antitrypsin protein (AAT) augmentation is a prescribed therapy for severe, genetically determined, alpha-1 antitrypsin deficiency (AATD), a genetic basis for pulmonary emphysema. AAT, a predominant systemic inhibitor of neutrophil elastase thus far has not been shown to decrease elastin degradation in a significant number of patients on this therapy. The objective of this study was to compare levels of biomarkers of elastin degradation in plasma, bronchoalveolar lavage (BALF) fluid and urine before and after beginning AAT augmentation therapy in patients with AATD. Methods: Desmosine and isodesmosine (DI), which occur only in elastin, are amino acid cross-links in mature elastin. Levels of DI in body fluids measure degradation of elastin and can be measured more specifically by mass spectrometry. This method was used to measure DI levels in plasma, bronchoalveolar lavage fluid and urine in cohorts of severe AATD patients on augmentation, not on augmentation and before and after the initiation of augmentation therapy. Results: Statistically significant reductions in plasma DI and in BALF DI were demonstrated in AATD patients receiving intravenous (IV) augmentation therapy as compared with those not receiving it. Administration by aerosol also produced statistically significant reductions in levels of DI in BALF. Conclusions: Results indicate that the currently prescribed doses of AAT augmentation inhibit neutrophil elastase adequately to reduce elastin degradation, both systemically and in the lung per se. The currently prescribed doses did not reduce elastin degradation to control levels, which may be possible with higher doses.

Stable deuterium internal standard for the isotope-dilution LC–MS/MS analysis of elastin degradation

Chemical synthesis of the deuterium isotope desmosine-d4 has been achieved. This isotopic compound possesses all four deuterium atoms at the alkanyl carbons of the alkyl amino acid substitution in the desmosine molecule and is stable toward acid hydrolysis; this is required in the measurement of two crosslinking molecules, desmosine and isodesmosine, as biomarkers of elastic tissue degradation. The degradation of elastin occurs in several widely prevalent diseases. The synthesized desmosine-d₄ is used as the internal standard to develop an accurate and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry analysis, which can serve as a generalized method for an accurate analysis of desmosine and isodesmosine as biomarkers in many types of biological tissues involving elastin degradation.

The Effect of Secondhand Smoke Exposure on Markers of Elastin Degradation

BACKGROUND Tobacco smoke is a major risk factor in the development of COPD. Secondhand smoke (SHS) exposure is a known risk factor in asthma, bronchitis, and coronary artery disease. Elastin is a recognized target for injury in COPD, and the amino acids desmosine and isodesmosine (D/I), which are specific for elastin degradation, are elevated in COPD. This study determined whether exposure to SHS affects elastin degradation in asymptomatic individuals. METHODS Two cohorts of asymptomatic individuals without evidence of respiratory or circulatory disease, exposed to SHS, were studied. Both cohorts comprised normal nonsmokers, active smokers, and those exposed to SHS. D/I were measured in plasma and quantified by high-performance liquid chromatography and tandem mass spectrometry by published methods. Plasma cotinine, a metabolite of nicotine, was also measured. RESULTS In each cohort, the levels of D/I in plasma were statistically significantly higher in secondhand-smoke-exposed subjects than in the normal nonexposed subjects. Smokers had the highest levels of D/I but their levels were not statistically significantly higher than those of the secondhand-smoke-exposed. Cotinine levels were elevated in secondhand-smoke-exposed subjects and active smokers but not in most nonsmoking control subjects. CONCLUSIONS Results indicate a tissue matrix effect of degradation of body elastin from SHS exposure and possible lung structure injury, which may result in COPD. Long-term studies of individuals exposed to SHS for the development of COPD are warranted.

Synergistic Effect of Hydrogen Peroxide and Elastase on Elastic Fiber Injury In Vitro

This laboratory has previously shown that hyperoxia enhances airspace enlargement in a hamster model of elastase-induced emphysema. To further understand the mechanism responsible for this finding, the effect of oxidants on elastase activity was studied in vitro, using a radiolabeled elastic fiber matrix derived from rat pleural mesothelial cells. Matrix samples were treated with either 0.1%, 1%, 3%, or 10% hydrogen peroxide (H2O2) for 1 hr, then incubated with 1.0 μg/ml porcine pancreatic elastase for 2 hrs. Radioactivity released from the matrix was used as a measure of elastolysis. Results indicate that sequential exposure to H2O2 and elastase markedly enhanced elastolysis compared to enzyme treatment alone. A 22% increase in elastolysis was seen with 0.1% H2O2 (325 vs. 396 cpm; P < 0.05), whereas samples pretreated with 1%, 3%, and 10% H2O2 showed increases of 53% (274 vs. 420 cpm; P < 0.05), 71% (381 vs. 653 cpm; P < 0.01), and 38% (322 vs. 443 cpm; P < 0.01), respectively. Exposure to various concentrations of H2O2 alone (0.1% to 10%) produced only minimal elastolysis (<20 cpm). However, 1% H2O2 was capable of degrading peptide-free desmosine and isodesmosine, suggesting that exposure to this oxidant may reduce the stability of the elastic fiber matrix. With regard to lung diseases such as emphysema, H2O2 and other oxidants derived from inflammatory cells or the environment could possibly act as priming agents for elastase-mediated breakdown of elastic fibers, resulting in amplification of lung injury.

Short-term cigarette smoke exposure potentiates endotoxin-induced pulmonary inflammation.

Long-term cigarette smoke exposure is associated with chronic obstructive pulmonary disease. However, the effects of short-term smoke inhalation are less clear, because it may adversely affect the lung only if underlying disease is present. To test this hypothesis, Syrian hamsters were passively exposed to cigarette smoke for 2 hours per day over a period of 3 days either before or after intratracheal instillation of low-dose (20 μg) Escherichia coli endotoxin. The results indicate that short-term smoke exposure can potentiate endotoxin-induced lung inflammation. They also suggest that nonsmokers with underlying lung disease may be particularly vulnerable to the adverse effects of second-hand smoke.

The Effect of Alpha-1 Proteinase Inhibitor on Biomarkers of Elastin Degradation in Alpha-1 Antitrypsin Deficiency: An Analysis of the RAPID/RAPID Extension Trials

The RAPID (NCT00261833; N=180) and RAPID Extension (NCT00670007; N=140) trials demonstrated significantly reduced lung density decline in patients with alpha-1 antitrypsin deficiency (AATD) receiving alpha-1 proteinase inhibitor (A1PI) versus placebo. Desmosine and isodesmosine (DES/IDES) are unique crosslinkers of mature elastin fibers and are utilized as measures of elastin degradation. The aim of this post-hoc study was to determine the effect of A1PI therapy on DES/IDES levels in patients from RAPID/RAPID Extension. Plasma levels of DES/IDES were measured using high-performance liquid chromatography and tandem mass spectrometry. Correlation between changes in DES/IDES levels and computed tomography (CT) lung density decline was assessed. Analysis showed that DES/IDES levels were significantly reduced versus baseline in patients receiving A1PI at all time points, from month 3 through month 48. A significant increase from baseline in DES/IDES was observed with placebo at month 24 (n=54; 0.016; p=0.018). DES/IDES change from baseline was significantly different with A1PI versus placebo at months 3 (-0.021; 95% confidence interval [CI] -0.037, 0.004; p=0.026), 12 (-0.040; 95% CI -0.055, 0.025; p<0.001), and 24 (-0.052; 95% CI -0.070, 0.034; p<0.001). Placebo patients started A1PI therapy at month 24 and showed significant reductions in plasma DES/IDES at months 36 (p<0.001) and 48 (p<0.001). Reduced elastin degradation was associated with slower lung density decline (p=0.005), correlating a chemical index of therapy with an anatomical index by CT. In conclusion, A1PI therapy reduced elastin degradation, including pulmonary elastin, in patients with AATD. These data support using DES/IDES levels as biomarkers to monitor emphysema progression and treatment response.