Enrichment and analysis of desmosine and isodesmosine in biological fluids

doi:10.1016/0378-4347(95)00092-W

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 668, Issue 2, 23 June 1995, Pages 199-207

https://doi.org/10.1016/0378-4347(95)00092-W Get rights and content

Abstract

A method has been developed for the enrichment and analysis of the elastin crosslinks, desmosine and isodesmosine, in biological fluids and tissues. It is adapted from published methods, offering improved recovery, sensitivity, resolution, and speed of analysis. Samples were hydrolyzed in 6 M HCl, after which the desmosines were enriched by CF1 cellulose chromatography and analyzed by HPLC with a C₁₈ column. Isodesmosine and desmosine were quantitated based on absorbance at 275 nm, with a limit of detection of approximately 30 pmol and recovery of approximately 66% in urine. Their t_R values on our HPLC system were approximately 9 and 12 min, respectively. This method was used to evaluate the daily and weekly variation in the concentrations of desmosine and isodesmosine in human urine. The results suggest that this method can be used to process large numbers of biological samples for analysis of desmosine and isodesmosine.

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Cited by (21)

Quantification of free and total desmosine and isodesmosine in human urine by liquid chromatography tandem mass spectrometry: A comparison of the surrogate-analyte and the surrogate-matrix approach for quantitation
2014, Journal of Chromatography A
Citation Excerpt :
Among the different biomarkers under study, desmosine (DES) and isodesmosine (IDS), two positional isomers that crosslink tropoelastin fibers in elastin, have been extensively investigated [4,5]. Initially, DES and IDS measurements in biological samples, particularly urine, relied on immunological techniques such as radioimmunoassay [6,7] or ELISA [8,9] as well as on spectrophotometric methods [10,11], all of them with limited selectivity and sensitivity [12,13]. Progressively, these methods have been replaced by more selective and sensitive methods such as capillary electrophoresis-laser induced fluorescence or liquid chromatography–mass spectrometry (LC–MS) [14–17].
In spite of the data suggesting the potential of urinary desmosine (DES) and isodesmosine (IDS) as biomarkers for elevated lung elastic fiber turnover, further validation in large-scale studies of COPD populations, as well as the analysis of longitudinal samples is required. Validated analytical methods that allow the accurate and precise quantification of DES and IDS in human urine are mandatory in order to properly evaluate the outcome of such clinical studies. In this work, we present the development and full validation of two methods that allow DES and IDS measurement in human urine, one for the free and one for the total (free + peptide-bound) forms. To this end we compared the two principle approaches that are used for the absolute quantification of endogenous compounds in biological samples, analysis against calibrators containing authentic analyte in surrogate matrix or containing surrogate analyte in authentic matrix. The validated methods were employed for the analysis of a small set of samples including healthy never-smokers, healthy current-smokers and COPD patients. This is the first time that the analysis of urinary free DES, free IDS, total DES, and total IDS has been fully validated and that the surrogate analyte approach has been evaluated for their quantification in biological samples. Results indicate that the presented methods have the necessary quality and level of validation to assess the potential of urinary DES and IDS levels as biomarkers for the progression of COPD and the effect of therapeutic interventions.
Stable deuterium internal standard for the isotope-dilution LC-MS/MS analysis of elastin degradation
2013, Analytical Biochemistry
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.
Micellar Electrokinetic Chromatography with Laser Induced Detection and liquid chromatography tandem mass-spectrometry-based desmosine assays in urine of patients with Chronic Obstructive Pulmonary Disease: A comparative analysis
2012, Journal of Chromatography A
Citation Excerpt :
On the other hand, the high number of different techniques developed by several laboratories around the world for their screening and quantification in a variety of fluids, reflects a growing interest in Des as an end-point in clinical trials. These techniques span from Radio Immuno Assay (RIA) [8–11] to High Performance Liquid Chromatography (HPLC) [12–16] and capillary electrophoresis (CE) [17]. In particular, the high sensitivity of Micellar Electrokinetic Chromatography with Laser Induced Fluorescence detection (MEKC-LIF), has proved successful in detecting these cross-links for the first time in serum and sputum as well [18].
Evidences accumulated over the past years that desmosines could be attractive indicators of elastic fibre degradation in Chronic Obstructive Pulmonary Disease have raised substantial interest with the development of reliable assays to measure their concentration in body fluids. It is a firm belief of researchers working in this field that accurate assessment of desmosine concentration would improve the understanding of elastin metabolism disorders and allow these cross-links to become a useful tool in the diagnosis and clinical management of these diseases. From among the variety of techniques available on the market, HPLC; CE and LC–MS have proved to be successful tools for measuring desmosines in biological fluids. However, differences in the analytical performance of methods may hinder the comparability of data, thus limiting the analytical strength and clinical utility of methods themselves. To address the relative contribution of different factors to the exact quantification of desmosines, the full potential of MEKC-LIF and LC–MS, the two systems that better than others offer more selective and sensitive detection for desmosine analysis, was studied on 56 urine samples. The results of this systematic comparative study underline the significant benefits of LC–MS over MEKC-LIF in terms of precision and sensitivity. Nevertheless, MEKC-LIF could be an attractive alternative in routine laboratories lacking the LC–MS instrumentation and skills to run these methods.
Quantitation of desmosine and isodesmosine in urine, plasma, and sputum by LC-MS/MS as biomarkers for elastin degradation
2011, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
COPD currently affects over 18 million Americans and is the fourth leading cause of death in the US. Various techniques including RIA [16,17], HPLC [18–20], and capillary zone electrophoresis [21,22] have been utilized for the analysis of urinary DES and IDS. We have developed a more specific and sensitive LC–MS analysis, which can measure DES and IDS in plasma, urine, and for the first time in sputum.
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 6 N 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.
Determination of desmosine in bronchoalveolar lavage fluids by time-resolved fluoroimmunoassay
2008, Clinica Chimica Acta
Citation Excerpt :
Although HPLC methods have excellent reproducibility, they have low sensitivity, and the desmosine peak is not sufficiently separated from the contaminant peaks. Hence, HPLC methods for determining desmosine in body fluids have generally required pre-treatment steps to remove contaminants and to enrich the analyte [28,29]. The recovery of desmosine in the pre-treatment step is generally low.
Urinary excretion of desmosine has been reported to be increased in patients with pulmonary fibrosis; however, several investigators have pointed out that measuring urinary desmosine is not a very useful indicator of lung wall destruction. We developed a sensitive time-resolved fluoroimmunoassay (TR-FIA) to identify trace amounts of desmosine in bronchoalveolar lavage fluid (BALF), and applied this method to analyze BALF samples from healthy subjects and patients with interstitial lung diseases.
In the proposed TR-FIA, a polystyrene strip was coated with desmosine-conjugated gelatin. The strip was then incubated with rabbit anti-desmosine antibody and the test solution. The desmosine bound to the solid phase and free desmosine in the sample or standard solution were allowed to compete to bind to the anti-desmosine. The solid-phase antibody was detected by Eu-complex conjugated anti-rabbit IgG.
The detectable limit of desmosine was 50 fmol/ml in the TR-FIA developed in this study. TR-FIA showed low cross-reactivity against amino acids. BALF desmosine levels were significantly higher in patients with idiopathic pulmonary fibrosis and sarcoidosis compared with healthy subjects.
Desmosine levels in BALF may be useful to investigate lung disease.
Effect of L-fucose and fucose-rich polysaccharides on elastin biosynthesis, in vivo and in vitro
2004, Biomedicine and Pharmacotherapy
With increasing age elastic fibres in human skin are progressively lysed and skin elasticity is also decreasing. Still there is an age-dependent increase of elastic fibre surface density, mostly due to an alteration of the fibres. The present experiments were undertaken to explore if L-fucose and fucose-rich polysaccharides (FROP-s) could influence elastin biosynthesis. We show here, that topical application of a fucose-containing preparation to the skin of hairless rats increased after 4 weeks the elastic fibre surface density by about 40%, shown by quantitative morphology. Using human skin fibroblasts in explant cultures, the addition of L-fucose or of FROP-3 increased the biosynthesis of immunoprecipitable tropoelastin by about 40%. No increase was found however of desmosine–isodesmosine in skin explant cultures after 72 h of incubation. The effect of L-fucose and FROP-3 on the biosynthesis of collagen and non-collagen proteins excreted by the skin explant cultures was also investigated. L-Fucose, but not FROP-3, decreased collagen biosynthesis but both increased non-collagen protein biosynthesis. These results show that L-fucose and FROP-3 stimulate tropoelastin biosynthesis in vitro, and elastic fibre formation in vivo. This stimulation concerns also several non-collagen proteins secreted by skin explant cultures. Elastic fibre formation necessitates the simultaneous synthesis of several microfibrillar glycoproteins as well as of tropoelastin. The increased elastic fibre density in the in vivo experiments suggests that this is indeed achieved by L-fucose and FROP-3, further demonstrating their efficiency in the control of age-dependent modifications of connective tissues in general and of skin in particular.

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Regular paperEnrichment and analysis of desmosine and isodesmosine in biological fluids

Abstract

Chest

Metabolism

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Ann. Rev. Med.

Am. Rev. Respir. Dis.

Am. Rev. Respir. Dis.

Am. Rev. Respir. Dis.

Regular paper
Enrichment and analysis of desmosine and isodesmosine in biological fluids