LC-MS/MS quantitation of elastin crosslinker desmosines and histological analysis of skin aging characteristics in mice.

Elastic fibers consist of an insoluble inner core of elastin, which confers elasticity and resilience to vertebral organs and tissues. Desmosine (DES) and isodesmosine (IDES) are potential biomarkers of pathologies that lead to decreased elastin turnover. Mice are commonly used in research to mimic humans because of their similar genetics, physiology, and organ systems. The present study thus used senescent accelerated prone (SAMP10) and senescent accelerated resistant (SAMR1) mice to examine the connection between aging and histological or biomolecular changes. Mice were divided into three groups: SAMP10 fed a control diet (CD), SAMP10 fed a high-fat diet (HFD), and SAMR1 fed a CD. The percent liver to total body weight ratio (%LW/BW), desmosines (DESs or DES/IDES) content, and histological alterations in skin samples were evaluated. DESs were quantified using an isotope-dilution liquid chromatography-tandem mass spectrometry method with isodesmosine-13C3,15N1 as the internal standard (ISTD). The assays were repeatable, reproducible, and accurate, with %CV values ≤ (1.90, 1.77, and 3.03), ISTD area %RSD of (1.54, 0.92, and 1.13), and %AC of (99.02 ± 1.86, 101.00 ± 2.30, and 101.30 ± 2.90) for the calibrations (equimolar DES/IDES, DES, and IDES, respectively). The average DESs content per dry-weight abdominal skin and %LW/BW were similar between the three groups. Histological analyses revealed elastin fibers in five randomly selected samples. The epidermis and dermal white adipose tissue layers were thicker in SAMP10 mice than SAMR1 mice. Thus, characteristic signs of aging in SAMP10 and SAMR1 mice could not be differentiated based on measurement of DESs content of the skin or %LW/BW, but aging could be differentiated based on microscopic analysis of histological changes in the skin components of SAMP10 and SAMR1 mice.

IsoChichibabin desmosine-13C3,15N1 synthesis and quantitative LC-MS/MS analysis of desmosine and isodesmosine in human skin.

Desmosine and isodesmosine are crosslinking amino acids of elastin, which is an essential component of the dermal extracellular matrix protein. Quantitative analysis of crosslinker desmosines in human skin dermis has not been fully achieved due to the insoluble nature of elastin protein. In the present study, chemical synthesis of isotopically labeled desmosine, desmosine-13C3,15N1, was carried out via isoChichibabin pyridinium synthesis starting from corresponding isotopically labeled amino acids. Isotope-dilution LC-MS/MS analysis of desmosine and isodesmosine utilizing synthetic desmosine-13C3,15N1 enabled the quantitative analysis of desmosines in human skin for the first time. Thus, ca. 1.43 µg of desmosines was detected from analysis of 1 mg of dry human skin.

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.

The Pattern of Elastic Fiber Breakdown in Bleomycin-Induced Pulmonary Fibrosis May Reflect Microarchitectural Changes.

INTRODUCTION: Desmosine and isodesmosine (DID) are unique elastin crosslinks that may serve as biomarkers for elastic fiber degradation in chronic obstructive pulmonary disease. Previously, our laboratory found that the ratio of free to peptide-bound DID in bronchoalveolar lavage fluid (BALF) showed a significant positive correlation with the extent of airspace enlargement in an elastase model of pulmonary emphysema. To further evaluate this hypothesis, our laboratory measured this ratio in a bleomycin (BLM) model of pulmonary fibrosis, which involved different microarchitectural changes than those associated with pulmonary emphysema. METHODS: Syrian hamsters were instilled intratracheally with 1.0 unit BLM in 0.2 ml of normal saline (controls received the vehicle alone), and BALF was analyzed for both free and total DID, using a combination of liquid chromatography and tandem mass spectrometry. RESULTS: Total BALF DID was significantly increased in hamsters receiving BLM at 1 week post-treatment (92 vs 13 pg/ml; p < 0.001), consistent with elastic fiber degradation. However, in contrast to elastase-induced emphysema, free/bound DID was lower in BLM-treated animals compared to controls at both 1 week (0.76 vs 0.84) and 2 weeks post-treatment (0.69 vs 0.86), though the differences were not statistically significant. CONCLUSIONS: These results indicate that it may be possible to identify specific pulmonary microarchitecture changes, based on the ratio of free to peptide-bound DID. It is speculated that the proportionate decrease in free DID in BLM-induced fibrosis may be due to preservation of intact elastic fibers as the lung injury progresses.

Fingerprinting desmosine-containing elastin peptides.

Elastin is a vital protein of the extracellular matrix of jawed vertebrates and provides elasticity to numerous tissues. It is secreted in the form of its soluble precursor tropoelastin, which is subsequently cross-linked in the course of the elastic fiber assembly. The process involves the formation of the two tetrafunctional amino acids desmosine (DES) and isodesmosine (IDES), which are unique to elastin. The resulting high degree of cross-linking confers remarkable properties, including mechanical integrity, insolubility, and long-term stability to the protein. These characteristics hinder the structural elucidation of mature elastin. However, MS(2) data of linear and cross-linked peptides released by proteolysis can provide indirect insights into the structure of elastin. In this study, we performed energy-resolved collision-induced dissociation experiments of DES, IDES, their derivatives, and DES-/IDES-containing peptides to determine characteristic product ions. It was found that all investigated compounds yielded the same product ion clusters at elevated collision energies. Elemental composition determination using the exact masses of these ions revealed molecular formulas of the type CxHyN, suggesting that the pyridinium core of DES/IDES remains intact even at relatively high collision energies. The finding of these specific product ions enabled the development of a similarity-based scoring algorithm that was successfully applied on LC-MS/MS data of bovine elastin digests for the identification of DES-/IDES-cross-linked peptides. This approach facilitates the straightforward investigation of native cross-links in elastin.

Electron transfer and collision induced dissociation of non-derivatized and derivatized desmosine and isodesmosine.

Electron transfer dissociation (ETD) has attracted increasing interest due to its complementarity to collision-induced dissociation (CID). ETD allows the direct localization of labile post-translational modifications, which is of main interest in proteomics where differences and similarities between ETD and CID have been widely studied. However, due to the fact that ETD requires precursor ions to carry at least two charges, little is known about differences in ETD and CID of small molecules such as metabolites. In this work, ETD and CID of desmosine (DES) and isodesmosine (IDS), two isomers that due to the presence of a pyridinium group can carry two charges after protonation, are studied and compared. In addition, the influence of DES/IDS derivatization with propionic anhydride and polyethyleneglycol (PEG) reagents on ETD and CID was studied, since this is a common strategy to increase sensitivity and to facilitate the analysis by reversed-phase chromatography. Clear differences between ETD and CID of non-derivatized and derivatized-DES/IDS were observed. While CID is mainly attributable to charge-directed fragmentation, ETD is initiated by the generation of a hydrogen atom at the initial protonation site and its subsequent transfer to the pyridinium ring of DES/IDS. These differences are reflected in the generation of complex CID spectra dominated by the loss of small, noninformative molecules (NH(3), CO, H(2)O), while ETD spectra are simpler and dominated by characteristic side-chain losses. This constitutes a potential advantage of ETD in comparison to CID when employed for the targeted analysis of DES/IDS in biological samples.

Cation exchange HPLC analysis of desmosines in elastin hydrolysates.

Desmosine crosslinks are responsible for the elastic properties of connective tissues in lungs and cardiovascular system and are often compromised in disease states. We developed a new, fast, and simple cation exchange HPLC assay for the analysis of desmosine and isodesmosine in animal elastin. The method was validated by determining linearity, accuracy, precision, and desmosines stability and was applied to measure levels of desmosines in porcine and murine organs. The detection and quantification limits were 2 and 4 pmol, respectively. The run-time was 8 min. Our cation exchange column does not separate desmosine and isodesmosine, but their level can be quantified from absorbance at different wavelengths. Using this assay, we found that desmosines levels were significantly lower in elastin isolated from various organs of immunodeficient severe combined immunodeficiency mice compared with wild-type animals. We also found that desmosines levels were lower in lung elastin isolated from hyperhomocysteinemic Pcft(-/-) mice deficient in intestinal folate transport compared with wild-type Pcft(+/+) animals.

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.

Development of a robust LC-MS/MS method for determination of desmosine and isodesmosine in human urine.

Desmosine (DES) and isodesmosine (IDES) are both pyridinium amino acid isomers that serve as cross-linking molecules binding the polymeric chains of amino acids into elastin. Found in urine, they are markers for the degradation of elastin which occurs in chronic obstructive pulmonary disease (COPD). In this study, a robust method using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) with selected reaction monitoring (SRM) mode was developed for the analysis of DES and IDES in human urine. Pyridylethyl-cysteine (PE-Cys) as internal standard (I.S.) was employed for the quantification of DES and IDES. The analytes and I.S. were extracted by solid-phase extraction with Oasis MCX cartridges and separated on an AccQ-Tag Ultra column. The assay was accurate (-6.8% to 14.5%) and precise (2.8% to 13.8%) within the concentration range of 1 to 250 pmol/mL. Moreover, the recovery and stability (working/ I.S. solution, urine samples with added elastin, and pretreated sample) was investigated, and these parameters were found acceptable. The UPLC-MS/MS method was validated and had good reproducibility and stability for the quantification of DES and IDES, which requires only 100 mL of human urine. This assay will be a useful means for measuring DES and IDES levels in urine with robustness and characterizing patients with COPD.

Separation and measurement of desmosine and isodesmosine in vascular tissue hydrolysates by micellar electrokinetic capillary chromatography with a mixed micelle system.

A micellar electrokinetic capillary chromatography (MEKC) method with a mixed micelle system for separation and measurement of desmosine (DES) and isodesmosine (IDES) in vascular tissue hydrolysates is described. The mixed micelle system was composed of a zwitterionic surfactant named 3-(N,N-dimethylhexadecylammonium)propanesulfonate (PAPS) and a nonionic surfactant polyethylene glycol dodecyl ether (Brij 35). By using 50 mM Tris-H(3)PO(4) (pH 2.5) containing 40 mM PAPS and 0.5% (m/v) Brij-35 as the optimal running buffer, DES and IDES were baseline separated from each other and from other hydrolyzed components of the vascular tissue. The limit of quantitation (LOQ) for DES and IDES were 3.00 x 10(-6)mol/L and 2.75 x 10(-6)mol/L, respectively. The relative standard deviation (RSD) of migration times and corrected peak area in terms of the inter-day and the intra-day repeatability were less than 1.7%. Hydrolysate samples of vascular tissues of rats were analyzed with the MEKC method with satisfied results.

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.

Biochemical characterization of collagen in alveolar mucosa and attached gingiva of pig.

Alveolar mucosa and attached gingiva are two continuous but functionally distinct connective tissues covering alveolar bone of the jaw. In this study, the major matrix component of these tissues, collagen, was biochemically characterized and compared. The tissues were obtained from mature pigs and analyzed for collagen content, amino acid composition, collagen types, collagen cross-linking, and gene expression. We found that alveolar mucosa is primarily composed of fibrillar collagens and the collagen content is higher than attached gingiva. The content of type III relative to type I collagen was higher in alveolar mucosa when compared with attached gingiva. The collagen cross-linking pattern also was distinct between the two tissues demonstrating that alveolar mucosa contained fewer reducible cross-links but more non-reducible cross-links in comparison to attached gingiva. The mRNA expression level of type I collagen in alveolar mucosa was significantly lower than that of attached gingiva. These results indicate that alveolar mucosa is a fibrillar collagen-rich tissue and, in comparison to gingival tissue, re-models slowly.

Progress in the methodological strategies for the detection in real samples of desmosine and isodesmosine, two biological markers of elastin degradation.

Desmosines are crosslinking amino acids unique to mature elastin in humans. Owing to this unicity, they have been discussed as potentially attractive indicators of connective tissue disorders whose clinical manifestations are mostly the result of elastin degradation. This review covers advances in immunochemical, chromatographic, and electrophoretic procedures applied in the last 25 years to detect and quantitate these crosslinksin a variety of biological samples. Recent applications of CE with LIF detection (CE-LIF) for investigating the content of desmosines in different fluids will also be discussed.

Use of a proteomics approach to identify favourable conditions for production of good quality lambskin leather.

It is necessary to understand the changes that occur during the initial processing of lamb skins, because these will affect the final quality of the leather. The types of collagen, their macro and micro structures, the presence of proteins other than collagens, and the quantity and the type of proteoglycans, all have a profound effect on the quality of leather. Proteins isolated from untreated or raw sheep skin and from pickled skin (skins treated with sodium sulfide and lime followed by bating with enzymes, then preserved in sodium chloride and sulfuric acid) were significantly different when analysed by use of 2D gel electrophoresis and mass spectrometry. Agarose gel electrophoresis with a very sensitive sequential staining procedure has been used to identify the glycosaminoglycans present in raw and treated skin and their impact on quality of leather. Results showed that effective removal of proteoglycans acting as inter-fibrillar adhesives of collagen fibrils seemed to improve leather quality. Removal of these molecules not only opens up the fibre structure of the skin but may also be important in wool removal. The presence of elastin, which imparts elastic properties to skin, is of significant importance to tanners. The amino acids desmosine and isodesmosine, found exclusively in elastin, were quantitatively analysed to assess the role of elastin in leather quality.

How a test for elastic fiber breakdown products in sputum could speed development of a treatment for pulmonary emphysema.

Pulmonary emphysema is a devastating disease for which there is no effective treatment. The development of therapeutic agents for this disorder has been hampered by the lack of clinical or biochemical tests which can rapidly evaluate drug efficacy. Since emphysema is associated with degradation of elastic fibers, the authors propose measuring the content of the elastin-specific amino acids, desmosine and isodesmosine, in sputum as a more immediate means of monitoring therapeutic interventions. Sputum samples would be chemically degraded to separate the component amino acids of elastic fibers, then measured for the total quantity of desmosine and isodesmosine, using any one of a number of established methods for quantifying these compounds, including radioimmunoassay, chromatography, or mass spectrometry. Such techniques allow for detection of nanogram quantities of desmosine and isodesmosine, and the procurement of ample amounts of induced sputum from the lower respiratory tract should improve the chances of detecting these amino acids. If proven valid, such a test could serve as a convenient marker for assessing lung injury in pulmonary emphysema, thereby facilitating rapid evaluation of new forms of treatment for this disease. The test might also prove to be a useful screening procedure for persons who smoke or otherwise have a greater than normal risk of developing emphysema.

LC/ESI-MS analysis of two elastin cross-links, desmosine and isodesmosine, and their radiation-induced degradation products.

In this work, the effect of Fenton reaction on two elastin cross-linked amino acids, desmosine (DES) and isodesmosine (IDE), in the absence or presence of different wavelength radiations generated from artificial sources has been evaluated using LC/ESI-MS. Irradiation as well as incubation of DES or IDE solutions in the presence of Fe(2+) and H(2)O(2) resulted in products with m/z 497.1 and 481.1 for [M+H](+). A strongly dose-dependent degradation of both amino acids was observed upon exposure to UVB at doses ranging from 0 to 3 J/cm(2) and a moderate dose-dependent degradation upon exposure to UVA at doses 10 times higher than that of UVB. A significant time-dependent degradation of DES and IDE was also observed upon exposure of these amino acids to a lamp emitting visible light similar to sunlight. Exposure of both amino acids to IR radiation (520 W) for 8 h did not cause significant degradation.

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.

Quantification of elastin cross-linking amino acids, desmosine and isodesmosine, in hydrolysates of rat lung by ion-pair liquid chromatography-mass spectrometry.

The elastin cross-linking amino acids, desmosine (DES) and isodesmosine (IDE), in hydrolysates of rat lungs were quantified by ion-pair liquid chromatography-electrospray mass spectrometry. The column was a 2.0 mm i.d. x 150 mm Develosil UG3 (ODS) with a mobile phase A of 7 mM pentafluoropropionic anhydride (PFPA) using ultrapure water as the ion-pair reagent, and a mobile phase B of 7 mM PFPA in 80% methanol. The retention times of IDE and DES were 25.5 and 26.6 min, respectively. The mean concentrations of IDE and DES in the lung were 191.6+/-54.5 nmol/g lung (dry tissue) (+/-SD) and 184.0+/-39.3 nmol/g lung, respectively, and the IDE/DES ratio was 1.04, in Wistar Kyoto rats. Our results indicate that ion-pair liquid chromatography-mass spectrometry is a useful procedure for quantitation of DES and IDE in hydrolysates of rat lung.

The changes in crosslink contents in tissues after formalin fixation.

The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks.