by Dr Jody M.¦W. van den Ouweland and Dr Rob Janssen
Desmosine is a promising biomarker for estimating elastin degradation activity in chronic obstructive pulmonary disease patients and provides a means to test the beneficial effects of therapeutic interventions. LC-MS/MS has emerged as a goldstandard method for accurate and sensitive measurement of desmosine in various body fluids, including plasma, urine, bronchoalveolar lavage fluid and sputum.
Chronic obstructive pulmonary disease (COPD) is one of the major health problems in the world, and currently the third leading cause of death by disease in the USA. COPD is a progressive lung disease defined by persistent airflow limitation predisposing the patient to exacerbations and serious illness. Distinct COPDphenotypes can be identified such as chronic bronchitis and emphysema. The disease is characterized by a low-grade inflammation and involves the release of enzymes that have the capacity to degrade the pulmonary elastic fibre network. Diagnosis is based on a combination of clinical symptoms and abnormalities in lung function tests. Chest radiology and arterial blood gas analysis are often used to establish disease severity. Validated lab tests that can be used in the management of COPD, however, are lacking. The current standard for determining COPD progression is through assessment of the decline of forced expiratory volume in one second (FEV1). As the rate of elastic fibre degradation is accelerated in COPD, matrix elastin degradation products may be effective biomarkers for estimating disease activity and to study the effect of therapeutic interventions . Elastin degradation is not unique for COPD and is also accelerated in several other chronic pulmonary conditions, including COPD, cystic fibrosis and tobacco use.
Principle of elastin synthesis and degradation
Elastin is a unique protein providing elasticity and resilience to dynamic organs, such as lungs and arteries and is thereby a basic requirement for both respiration and circulation. Elastin is synthesized in various cells which secrete the soluble precursor, monomer tropoelastin, into the extracellular matrix, which is then cross-linked mainly through formation of two amino acids, desmosine and isodesmosine (Dl), which are derived from the condensation of four lysine residues of elastin molecules by lysyl-oxidase (Fig. 1). The DI pyridinium ring has three allysyl side chains and one unaltered lysyl side chain (Fig. 2). Cross-linking transforms the soluble tropoelastin to the insoluble cross-linked mature elastin fibre. DI, as a cross-linker of elastin, gives elasticity to the tissue (Fig. 1). DI occurs only in mature elastin and its presence in body fluids is an indicator of degradation of mature elastic fibres .
Desmosine as a biomarker of elastin degradation
DI is one of the oldest discovered biomarkers and was developed in the 1960s, but the first time it was correlated to lung elastin content was in the 1980s. As the concentrations of DI in body fluids are extremely low, their precise and specific measurements have been a challenge. Initially, DI measurements in biological samples, particularly urine, relied on immunological techniques such as radioimmunoassay or ELISA as well as on spectrophotometric methods, all of them with limited selectivity and sensitivity, and inconsistencies in measured concentrations. Progressively, these methods have been replaced by more selective and sensitive methods such as capillary electrophoresis laser-induced fluorescence or liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowing measurement of DI in body fluids, including urine, plasma, bronchoalveolar lavage fluid and sputum . Moreover, LC-MS/MS has shown much better inter-method agreement than other assays.
LC-MS/MS measurement of desmosine in body fluids
It has shown to be possible with LC-MS/MS to accurately measure DI in body fluids, including urine, plasma, bronchoalveolar lavage fluid and sputum. The assay procedure for measuring total DI is rather laborious comprising three major steps including acid hydrolysis, solid phase extraction (SPE) with drying/resuspension, and LC-MS/MS. In brief, it starts with adding an equal volume of concentrated hydrochloric acid to plasma, urine or other body fluid including isotopically-labelled desmosine-d4 as internal standard, followed by a 24-hour incubation at 110|°C to liberate DI covalently bound to DI-containing peptides. Next, cellulose SPE is performed to extract total DI from plasma or urine after which the extract is dried and resuspended. Chromatographic separation of both isomers is achieved on a C18 column by addition of an ion-pairing reagent to the mobile phase, followed by selected reaction monitoring by mass spectrometry.
What was not anticipated were the many hurdles in the developmental process, taking years before the assay was ready to be used for clinical research in our hospital. First, the harsh acidic conditions used in sample preparation resulted in corrosion of stainless steel needles in the SPE manifold and in the dry-down heating block with consequent loss of peak signals. Second, discontinuation of critical SPE material by the manufacturer led to a long-lasting search for suitable alternatives. Finally, a twofold difference in measured concentrations of DI was observed when compared to data obtained from literature that could be traced back to an error in designation of the DI standard concen-tration by the supplier. Since then, our LC-MS/MS assay appears robust with performance of over 3000 analyses in various specimens and clinical application areas. The assay has a broad measuring range of 0.14–210|μg/L for DI enabling measurement in various body fluids.
Clinical application areas
We started our quest for an intervention to decelerate elastic fibre degradation. We studied the effect of vitamin|D administration on DI levels in COPD patients but did not find a favourable effect. From vitamin|D, we became interested in vitamin|K and were the first to demonstrate an inverse correlation between vitamin|K status and plasma DI levels . We found this association in patients with COPD and idiopathic pulmonary fibrosis (IPF) as well as in subjects using vitamin|K antagonists as anticoagulant medication. We are currently planning intervention trials in COPD to evaluate whether vitamin|K supplementation reduces DI levels. Elastic degradation accelerates during ageing and is particularly pronounced in COPD and IPF. Reference values for DI increase during ageing and have been established for non-smokers and smokers without lung diseases as well as for patients with COPD and IPF. DI levels appeared to be equally increased in IPF as in COPD . In cystic fibrosis patients, plasma DI correlated with lung function, exacerbation frequency and disease progression, suggesting that measuring DI levels in body fluids by LC-MS/MS may be an effective strategy of monitoring disease progression in cystic fibrosis patients .
A large study in 1177 COPD patients investigated the association between plasma DI and emphysema severity/progression, coronary artery calcium score and mortality . It was found that in COPD, excess elastin degradation relates to cardiovascular comorbidities, atherosclerosis, arterial stiffness, systemic inflammation and mortality, but not to emphysema or emphysema progression. The latter may be due to the heterogenicity of the study population including distinct COPD-phenotypes from chronic bronchitis to emphysema. Indeed, elastin is not only present in alveolar walls but also in airways and plasma DI does not therefore specifically reflect emphysema formation. This can well explain why plasma DI was not correlated with emphysema progression in this heterogenous COPD population. Accelerated elastin degradation could potentially contribute to both the pulmonary and extrapulmonary disease manifestations of COPD and may represent a mechanistic link between COPD and the increased risk of cardiovascular disease.
Plasma DI levels correlate with emphysema severity on CT scan in patients with the genetic disorder alpha-1 antitrypsin deficiency (AATD). These patients have insufficient or absent AAT to protect elastic fibres from degradation by proteases, in particular neutrophil elastase. Weekly administration of alpha-1 antitrypsin reduced plasma DI levels . Given that loss of lung parenchyma is irreversible, early initiation in subjects with AATD and elevated plasma DI levels may be an attractive strategy to prevent permanent lung function decline. A plausible reason why plasma DI was correlated with emphysema in AATD patients and not in a heterogenous group of COPD patients, is that AATD patients are a rather homogeneous group with a common predominant form of panlobular emphysema in the basal lung fields.
Finally, in a recent study in SARS-CoV-2 patients, we found impaired vitamin|K-dependent matrix-Gla-protein activation, as a measure of extrahepatic vitamin|K status, linked to accelerated elastic fibre degradation and premorbid vascular calcifications as measured by DI in plasma . We are currently planning intervention trials in COVID-19 patients to evaluate whether vitamin|K supplementation improves outcome of SARS-CoV-2 infections.
In conclusion, the detection and measurement of DI as a means to study elastin degradation has been used for almost 30|years; however, recent methodological advances by our group and others have aided DI detection, as the concentrations present in body fluids are extremely low.
Conflict of interest statement
JO and RJ are owners of Desmosine.com. RJ discloses application of a patent for vitamin|K status as a prognostic and therapeutic biomarker in COVID-19.
Jody M.W. van den Ouweland*1 PhD and Rob Janssen2 MD
1Department of laboratory Medicine, Canisius-Wilhelmina Hospital, 6532, SZ, Nijmegen, The Netherlands
2Department of Pulmonary Medicine, Canisius-Wilhelmina Hospital, 6532, SZ, Nijmegen, The Netherlands