The Therapeutic Potential of Hyaluronan in COPD.

Insights into the clinical course of COPD indicate the need for new therapies for this condition. The discovery of alpha-1 antitrypsin deficiency (AATD) led to the protease-antiprotease imbalance hypothesis, which was applied to COPD related to AATD as well as COPD not related to AATD. The discovery of AATD brought recognition to the importance of elastin fibers in maintaining lung matrix structure. Two cross-linking amino acids, desmosine and isodesmosine (DI), are unique to mature elastin and can serve as biomarkers of the degradation of elastin. The intravenous augmentation treatment and lung density in severe alpha-1 antitrypsin deficiency (RAPID) study shows a correlation of an anatomic index of COPD (on CT imaging) correlating with a chemical indicator of matrix injury in COPD, DI. The results suggest that preservation of lung elastin structure may slow the progression of COPD. Hyaluronan aerosol decreases the severity of elastase-induced emphysema in animals and has induced reductions in DI levels in preliminary human studies. Hyaluronan deserves further development as a therapy for COPD.

Desmosine and Isodesmosine as a Novel Biomarker for Pulmonary Arterial Hypertension: A Pilot Study.

Delayed diagnosis is common in patients with pulmonary arterial hypertension (PAH). Right-sided heart catheterization, the gold standard for diagnosis, is invasive and cannot be applied for routine screening. Some biomarkers have been looked into; however, due to the lack of a clear pathological mechanism linking the marker to PAH, the search for an ideal one is still ongoing. Elastin is a significant structural constituent of blood vessels. Its synthesis involves cross-linking of monomers by 2 amino acids, desmosine and isodesmosine (D&I). Being extremely stable, elastin undergoes little metabolic turnover in healthy individuals resulting in very low levels of D&I amino acids in the human plasma, urine, or sputum. We hypothesized that in PAH patients, the elastin turnover is high; which in turn should result in elevated levels of D&I in plasma and urine. Using mass spectrometry, plasma and urine levels of D&I were measured in 20 consecutive patients with PAH confirmed by cardiac catheterization. The levels were compared with 13 healthy controls. The mean level of total plasma D&I in patients with PAH was 0.47 ng/mL and in controls was 0.19 ng/mL (P = 0.001). The mean levels of total D&I in the urine of PAH patients was 20.55 mg/g creatinine and in controls was 12.78 mg/g creatinine (P = 0.005). The mean level of free D&I in the urine of PAH patients was 10.34 mg/g creatinine and in controls was 2.52 mg/g creatinine (P < 0.001). This is the first study highlighting that the serum and urine D&I has a potential to be a novel screening biomarker for patients with PAH. It paves the way for larger studies to analyze its role in assessing for disease severity and response to treatment.

Biomarkers in Alpha-1 Antitrypsin Deficiency Chronic Obstructive Pulmonary Disease.

Biomarkers of pathogenesis in chronic obstructive pulmonary disease (COPD) can significantly accelerate drug development. In COPD related to alpha-1 antitrypsin deficiency, the role of neutrophil elastase and its inhibition by alpha-1 antitrypsin protein focused interest on elastin degradation and the development of pulmonary emphysema. Amino acids desmosine and isodesmosine are unique cross-links in mature elastin fibers and can serve as biomarkers of elastin degradation when measured in body fluids. This review gives a perspective on what has been learned by the earliest measurements of desmosine and isodesmosine followed by later studies using methods of increased sensitivity and specificity and the meaning for developing new therapies. Also included are brief statements on the biomarkers fibrinogen, CC-16, and Aa-Val-360 in COPD.

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.

Synthesis and LC-MS/MS analysis of desmosine-CH2, a potential internal standard for the degraded elastin biomarker desmosine.

Desmosine-CH2, an analog of the elastic tissue degradation biomarker desmosine, can be regarded as a potential internal standard for precise quantification of desmosines by LC-MS/MS. In this study, the chemical synthesis of desmosine-CH2 was completed in 22% overall yield in five steps. The LC-MS/MS analysis of desmosine-CH2 was also achieved.

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.

Alpha-1 antitrypsin augmentation therapy and biomarkers of elastin degradation.

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.

Total synthesis of COPD biomarker desmosine that crosslinks elastin.

Desmosine, a crosslinking amino acid of elastin, is an attractive biomarker for diagnosis of chronic obstructive pulmonary disease (COPD). In this study, the first total synthesis of (+)-desmosine was achieved in 11% overall yield in 13 steps utilizing stepwise and regioselective Sonogashira cross-coupling reactions.

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.

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.

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.

Characterization of peptide fragments from lung elastin degradation in chronic obstructive pulmonary disease.

This study presents a method for detecting and characterizing peptides of elastin that result from lung matrix injury in chronic obstructive pulmonary disease (COPD). Lung elastin degradation was studied by two representative in vivo elastases, human neutrophil elastase (HNE) and macrophage metalloproteinase (MMP12). The resulting peptide mixtures were analyzed by high-performance liquid chromatography/electrospray tandem mass spectrometry (LC/MSMS) to characterize 40 elastin-derived peptides (EDPs), 24 from HNE and 16 from MMP12 digestions. The peptides constitute major EDPs that are solubilized by the enzymatic digestion. Using the selected reaction monitoring (SRM) from LC/MSMS analysis, the transition ions of the peptides were used to investigate the presence of the peptides in selected body fluids of chronic obstructive pulmonary disease (COPD) patients. Four peptides, GYPI, APGVGV, GLGAFPA, and VGVLPGVPT, were detected in plasma or sputum of some COPD patients but not in normal controls. A hexapeptide VGVAPG, which had been widely studied for its chemotactic activity for a possible pathogenic role in COPD, was not detected in lung EDPs by HNE or MMP12 digestion, but only by porcine pancreatic elastase (PPE) digestion. This study demonstrates a practical methodology to study peptides from matrix degradations in pulmonary disease and a means of investigating their pathogenesis.

The effect of tiotropium therapy on markers of elastin degradation in COPD.

BACKGROUND: Desmosine and Isodesmosine (D/I) are cross-linking amino acids which are present only in mature elastin. Changes in their concentration in body fluids indicate changes in elastin degradation and can be a reflection of tissue elastase activity. This study was undertaken to determine whether continuous therapy with the long-acting bronchodilator Tiotropium bromide (TTP) could result in reductions in D/I as measured by mass spectrometry in plasma, urine and sputum. METHODS: Twelve not currently smoking patients with chronic obstructive pulmonary disease (COPD), never on TTP, were selected for study. Levels of D/I, along with measurements of FVC, FEV1 and FEV1/FVC. were determined before starting TTP daily, and then one and two months after. RESULTS: D/I decreased in plasma (10 of 12 patients), in sputum all (12 of 12), and in the percentage of free D/I in urine (10 of 12). Most patients showed slight increases in FVC and FEV1 percent predicted over two months. CONCLUSION: The results are consistent with an effect of prolonged bronchodilitation by anti-cholinergic blockade to also result in reduced lung elastin degradation.

Measurements of desmosine and isodesmosine by mass spectrometry in COPD.

OBJECTIVES: Application of mass spectrometry (MS) for direct measurements of desmosine (D) and isodesmosine (I) in urine, plasma, and sputum as markers of elastin degradation in patients with alpha(1)-antitrypsin deficiency (AATD) and non-AATD-related COPD. BACKGROUND: In COPD patients, the lungs undergo elastin injury, which can be monitored by measurements of D and I in body fluids as specific markers of elastin degradation using the specificity and sensitivity of MS. METHODS: Acid hydrolysis of blood plasma, 24-h urine and sputum measurements, followed by chromatographic separation for mass spectrometric analysis. RESULTS: Each patient group had levels of plasma D and I that were statistically significantly higher than those of control subjects. AATD patients had higher levels than COPD patients with normal alpha(1)-antitrypsin (AAT) levels. Twenty-four-hour urine measurements demonstrated no significant difference in total levels of D and I among control subjects and patients but showed a free (unbound) concentration of D and I in urine, which was statistically significantly higher in patients with COPD with and without AAT. The D and I levels in the sputum of patients with AATD exceeded the levels in COPD patients with normal AAT levels. CONCLUSIONS: MS allows a sensitive and specific analysis of D and I in body fluids. The quantification of D and I in sputum, along with increases of D and I in plasma and an elevated free component of D and I in urine provide indexes that characterize patients with COPD and can be followed in relation to the course of the disease and/or therapy.

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 microg/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 microg/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.