The prognostic values of plasma desmosines, crosslinking molecules of elastic fibers, in the disease progression of Moyamoya disease.

Moyamoya disease (MMD) is a cerebrovascular disease which is characterized by the chronic progression of steno-occlusive changes at the terminal portion of internal carotid arteries and the development of "moyamoya vessels." Dysregulation of the extracellular matrix is regarded as a key pathophysiology underlying unique vascular remodeling. Here, we measured the concentration of elastin crosslinkers desmosine and isodesmosine in the plasma of MMD patients. We aimed to reveal its diagnostic values of desmosines in the progression of steno-occlusive lesions. The concentrations of plasma desmosines were determined by liquid chromatography-tandem mass spectrometry. The temporal profiles of steno-occlusive lesions on magnetic resonance angiography were retrospectively evaluated, and the correlation between the progression of steno-occlusive changes in intracranial arteries and plasma desmosines concentrations was further analyzed. Plasma desmosines were significantly higher in MMD patients with disease progression compared to MMD patients without disease progression. Also, the incidence of disease progression was higher in MMD patients with plasma desmosines levels over limit of quantitation (LOQ) than those with plasma desmosines levels below LOQ. In conclusion, plasma desmosines could be potential biomarkers to predict the progression of steno-occlusive changes in MMD patients.

LC-MS/MS analysis of elastin crosslinker desmosines and microscopic evaluation in clinical samples of patients with hypertrophy of ligamentum flavum.

Ligamentum flavum (LF) pathologies often lead to severe myelopathy or radiculopathy characterized by reduced elasticity, obvious thickening, or worsened ossification. Elastin endows critical mechanical properties to tissues and organs such as vertebrae and ligaments. Desmosine (DES) and isodesmosine (IDES) are crosslinkers of elastin monomers called tropoelastin. These crosslinkers are potential biomarkers of chronic obstructive pulmonary disease. As a biological diagnostic tool that supplements existing symptomatic, magnetic resonance imaging scanning or radiological imaging diagnostic measures for LF hypertrophy and associated pathologies, an isotope-dilution liquid chromatography-tandem mass spectrometry method with selected reaction monitoring mode for the quantitation of DESs in human plasma, urine, cerebrospinal fluid (CSF), and yellow ligamentum was investigated. Isotopically labeled IDES-13C3,15N1 was used as an internal standard (ISTD) for DES quantitation for the first time. The samples plus ISTD were hydrolyzed with 6 N hydrochloric acid. Analytes and ISTD were extracted using a solid phase extraction cellulose cartridge column. The assays were repeatable, reproducible, and accurate with % CV ≤ 7.7, ISTD area % RSD of 7.6, and % AC ≤ (101.2 ± 3.90) of the calibrations. The ligamentum samples gave the highest average DES/IDES content (2.38 µg/mg) on a dry-weight basis. A high percentage of the CSF samples showed almost no DESs. Urine and plasma samples of patients showed no significant difference from the control (p-value = 0.0519 and 0.5707, respectively). Microscopy of the yellow ligamentum samples revealed dark or blue-colored zones of elastin fibers that retained the hematoxylin dye and highly red-colored zones of collagen after counterstaining with van Gieson solution. Thus, we successfully developed a method for DES/IDES quantitation in clinical samples.

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.

Quantification of desmosine and isodesmosine using MALDI-ion trap tandem mass spectrometry.

Desmosine (Des) and isodesmosine (Isodes), cross-linking amino acids in the biomolecule elastin, may be used as biomarkers for various pathological conditions associated with elastin degradation. The current study presents a novel approach to quantify Des and Isodes using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS2) in a linear ion trap coupled to a vacuum MALDI source. MALDI-MS2 analyses of Des and Isodes are performed using stable-isotope-labeled desmosine d4 (labeled-Des) as an internal standard in different biological fluids, such as urine and serum. The method demonstrated linearity over two orders of magnitude with a detection limit of 0.02 ng/µL in both urine and serum without enrichment prior to mass spectrometry, and relative standard deviation of < 5%. The method is used to evaluate the time-dependent degradation of Des upon UV irradiation (254 nm) and found to be consistent with quantification by 1H NMR. This is the first characterized MALDI-MS2 method for quantification of Des and Isodes and illustrates the potential of MALDI-ion trap MS2 for effective quantification of biomolecules. The reported method represents improvement over current liquid chromatography-based methods with respect to analysis time and solvent consumption, while maintaining similar analytical characteristics. Graphical abstract ᅟ.

A new approach for quantitative characterization of hydrolytic action of proteases to elastin in leather manufacturing.

Leather biotechnology based on enzyme is one of the main directions toward clean technology in the leather manufacturing process. Proteins such as collagen, elastin, and keratin are important components in animal hides or skins, and proteases are most frequently used in the leather manufacturing process for the removal of interfibrillar substance and opening-up of collagen fiber instead of toxic chemicals. Elastin is an important and highly elastic structural protein in the animal hides or skins and significantly affects the properties of the final leather product. For improving the quality of leather product, thorough understanding of the mechanism of action of proteases on elastin is necessary. The action of proteases on elastin has been mostly studied either qualitatively by histological analysis or quantitatively based on substrate casein or stained substrates, such as congo red-elastin and Remazol Brilliant Blue R-elastin; however, the resulting products have not been accurately characterized and thus these methods are not up to the standard. Besides, controlling the hydrolytic action of proteases to elastin has been very difficult, and excessive hydrolytic action of protease damages the elastin, restricting the wide application of proteases in the leather manufacturing process. In order to quantitatively evaluate the hydrolytic action of proteases on elastin in a more accurate manner, in this study, a new method was established by determining the unique amino acid desmosine based on the covalently bonded elastin-desmosine conjugate. Quantitative analysis of desmosine was performed in liquor based on cowhides substrate, and qualitative characterization was accomplished by histological analysis of elastic fiber in hides using an optical microscope. The results of this study indicated that the newly developed method is sensitive, accurate, and reproducible. In addition, the unhairing trials also demonstrated the suitability of newly established method in the leather manufacturing process to evaluate the action of proteases on the elastin in animal hides or skins.

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.

Advances in Identifying Urine/Serum Biomarkers in Alpha-1 Antitrypsin Deficiency for More Personalized Future Treatment Strategies.

Alpha1-antitrypsin deficiency (AATD) is a genetic disorder characterized by reduced serum levels of alpha1-antitrypsin (AAT) and increased risk for developing both early-onset lung emphysema and chronic liver disease. Laboratory diagnosis of AATD is not just a matter of degree, although the AAT serum level is the most important determinant for risk of lung damage. While being a single-gene disease, the clinical phenotype of AATD is heterogeneous. The current standard of care for patients affected by AATD-associated pulmonary emphysema is replacement therapy with weekly i.v. infusions of pooled human purified plasma AAT. Although no treatment for liver disease caused by deposition of abnormal AAT in hepatocytes is available, innovative treatments for this condition are on the horizon. This article aims to provide a critical review of the methodological steps that have marked progress in the detection of indicators described in the literature as being "clinically significant" biomarkers of the disease. The development and routine use of specific biomarkers would help both in identifying which patients and when they are eligible for treatment as well as providing additional parameters for monitoring the disease.

[(13)C3,(15)N1]-labeled isodesmosine: A potential internal standard for LC-MS/MS analysis of desmosines in elastin degradation.

Isodesmosine and desmosine are crosslinking amino acids that are present only in elastin. They are useful biomarkers for the degradation of elastin, which occurs during the progression of chronic obstructive pulmonary disease (COPD) and related diseases. This Letter describes the synthesis of [(13)C3,(15)N1]-labeled isodesmosine, using Chichibabin pyridine synthesis as a key reaction. The labeled isodesmosine is a potential internal standard for the quantitative LC-MS/MS analysis of desmosines in elastin degradation.

From micellar electrokinetic chromatography to liquid chromatography-mass spectrometry: revisiting the way of analyzing human fluids for the search of desmosines, putative biomarkers of chronic obstructive pulmonary disease.

Desmosine (DES) and isodesmosine are two isomer amino acids unique-to-mature, cross-linked elastin. Based on this feature, they have been discussed as surrogate markers of chronic obstructive pulmonary disease, a disorder characterized by progressive degradation of lung elastin. Despite the development of numerous protocols, detection of DESs in body fluids is still considered to be technically challenging. In fact, owing to the minute concentration of these circulating cross-links, their accurate measurement may be provided only by sophisticated and sensitive techniques. Aim of this article is to present the "history" of the two techniques (MEKC and LC-MS) that, better than others, allowed scientists to "bring their best to the table" in this area. Both of them meet the criteria of (almost) complete automation of the procedure and of the use of more selective and sensitive detection systems. The substantial advantages in terms of precision and accuracy provided by such measurements suggest that the science of DESs is eventually catching up with its promise and the assumption that these candidate biomarkers can be associated to clinical variables holds true.

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.

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.

Extracts from Glycine max (soybean) induce elastin synthesis and inhibit elastase activity.

Elastic fibres are essential extracellular matrix components of the skin, contributing to its resilience and elasticity. In the course of skin ageing, elastin synthesis is reduced, and elastase activity is accelerated, resulting in skin sagging and reduced skin elasticity. Our studies show that non-denatured Glycine max (soybean) extracts induced elastin promoter activity, inhibited elastase activity and protected elastic fibres from degradation by exogenous elastases in vitro. Mouse and swine skins topically treated with soybean extracts showed enhanced elastic fibre network and increased desmosine content. Elastin expression was also augmented in human skin transplanted onto SCID mice in response to soy treatment. These data suggest that non-denatured soybean extracts may be used as skin care agents to reduce the signs of skin ageing.

Localization of lysyl oxidase in hen oviduct: implications in egg shell membrane formation and composition.

Lysyl oxidase activity was found in the isthmus (the membrane-forming region) of the hen's oviduct in a copper-rich region proximal to the shell gland. Desmosine and isodesmosine, cross-linking compounds associated with mature elastin, were found in hydrolysates of the shell membrane, confirming the necessity for lysyl oxidase in its biosynthesis. Shell membranes from hens fed a copper-deficient diet or a diet supplemented with beta-aminopropionitrile had a reduced content of desmosine and isodesmosine.

Elastin haploinsufficiency induces alternative aging processes in the aorta.

Elastin, the main component of elastic fibers, is synthesized only in early life and provides the blood vessels with their elastic properties. With aging, elastin is progressively degraded, leading to arterial enlargement, stiffening, and dysfunction. Also, elastin is a key regulator of vascular smooth muscle cell proliferation and migration during development since heterozygous mutations in its gene (Eln) are responsible for a severe obstructive vascular disease, supravalvular aortic stenosis, isolated or associated to Williams syndrome. Here, we have studied whether early elastin synthesis could also influence the aging processes, by comparing the structure and function of ascending aorta from 6- and 24-month-old Eln+/- and Eln+/+ mice. Eln+/- animals have high blood pressure and arteries with smaller diameters and more rigid walls containing additional although thinner elastic lamellas. Nevertheless, longevity of these animals is unaffected. In young adult Eln+/- mice, some features resemble vascular aging of wild-type animals: cardiac hypertrophy, loss of elasticity of the arterial wall through enhanced fragmentation of the elastic fibers, and extracellular matrix accumulation in the aortic wall, in particular in the intima. In Eln+/- animals, we also observed an age-dependent alteration of endothelial vasorelaxant function. On the contrary, Eln+/- mice were protected from several classical consequences of aging visible in aged Eln+/+ mice, such as arterial wall thickening and alteration of alpha(1)-adrenoceptor-mediated vasoconstriction. Our results suggest that early elastin expression and organization modify arterial aging through their impact on both vascular cell physiology and structure and mechanics of blood vessels.

Determination of desmosine in bronchoalveolar lavage fluids by time-resolved fluoroimmunoassay.

BACKGROUND: 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 analyse BALF samples from healthy subjects and patients with interstitial lung diseases. METHODS: 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. RESULTS: 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 fibrosis and sarcoidosis compared with healthy subjects. CONCLUSIONS: Desmosine levels in BALF may be useful to investigate lung disease.

Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model.

Although matrix metalloproteinases (MMPs) are expressed in abundance in arterial aneurysms, their contribution to arterial wall degeneration, dilation, and rupture has not been determined. We investigated MMP function in a rat model of aneurysm associated with arterial dilation, elastin loss, medial invasion by mononuclear inflammatory cells, and MMP upregulation. Rupture was correlated with increased gelatinase B (MMP-9) and activated gelatinase A (MMP-2). Syngeneic rat smooth muscle cells retrovirally transfected with tissue inhibitor of matrix metalloproteinases (TIMP)-1 cDNA (LTSN) or with the vector alone as a control (LXSN) were seeded onto the luminal surface of the vessels. The seeding of LTSN cells resulted in TIMP-1 local overexpression. The seeding with LTSN cells, but not LXSN cells, decreased MMP-9, activated MMP-2 and 28-kD caseinase and elastase activity, preserved elastin in the media, and prevented aneurysmal degeneration and rupture. We conclude that MMP overexpression is responsible for aneurysmal degeneration and rupture in this rat model and that local pharmacological blockade might be a reasonable strategy for controlling the formation of aneurysms in humans.

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.

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.