The biosynthesis of desmosine and isodesmosine. II. Incorporation of specifically labeled lyxine into desmosine and isodesmosine.
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Adolescent↗
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The estimation of elastin in fetal tissues by radioimmunoassay of isodesmosine.
A radioimmunoassay was developed for the determination of isodesmosine as the tetraacetyl derivative. Isodesmosine tetraacetate conjugated with bovine albumin was injected into rabbits which developed useful titers of antibodies after five months. The radioligand for the assay was prepared by acetylating isodesmosine with [3H] acetic anhydride. The bound ligand was separated from free ligand by coprecipitation with human gamma-globulin in 46% saturated ammonium sulfate solution. The sensitivity of the assay was 2 ng isodesmosine. The antiserum was specific for isodesmosine tetraacetate and only desmosine tetraacetate gave appreciable cross-reactivity (4%). The assay was found to be suitable for the accurate estimation of elastin in small samples (5 mg dry weight) of rat and ovine fetal lung tissue and for elastin degradation products in amniotic fluid (0.5 ml).
Amino Acids↗
Enrichment and analysis of desmosine and isodesmosine in biological fluids.
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 tR 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.
Animals↗
Photolysis of desmosine and isodesmosine by ultraviolet light.
1. Desmosine and isodesmosine were separated by ion-exchange and paper chromatography, after acid hydrolysis of purified elastin from beef ligamentum nuchae. The fractions obtained by ion-exchange chromatography were clearly mixtures of related compounds. The desmosine fraction could be resolved into seven compounds and the isodesmosine into four by paper chromatography. 2. Desmosine was maximally degraded by irradiation at 274 nm and isodesmosine at 285 nm. These wavelengths did not correspond to the absorption maxima of the cross links, but to shoulders of the main absorption peaks. 3. When irradiated at their optimum wavelengths, but at various pH, both desmosine and isodesmosine seemed quite stable at pH greater than 8.5. Between pH 8 and 5, the photolytic rate was maximum and decreased slightly at more acidic pH. Below pH 4.0, one of the products of photolysis was free lysine. 4. In analogy to the mechanism of the photolytic degradation of N-methyl pyridinium chloride, it appears that the (iso)desmosines were degraded via the formation of an open amino aldehyde, which was hydrolysed at acid pH to give free lysine and a substituted glutaconic aldehyde.
Amino Acids↗
High-performance liquid chromatographic quantitation of desmosine plus isodesmosine in elastin and whole tissue hydrolysates.
Quantitation of desmosine and isodesmosine, the major crosslinks in elastin, has been of interest because of their uniqueness and use as markers of that protein. Accurate measurement of these crosslinks may allow determination of elastin degradation in vivo and elastin content in tissues, obviating lengthy extraction procedures. We have developed a method of quantitating desmosine plus isodesmosine in hydrolysates of tissue and insoluble elastin using high-performance liquid chromatographic separation and absorbance detection that is rapid (21-35 min) and sensitive (accurate linearity from 100 pmol to 5 nmol). This method has been used to quantitate desmosines in elastin from bovine nuchal ligament and lung and in whole aorta from hamster. The ability to completely separate [3H]lysine from desmosine plus isodesmosine allows the method to be used to study incorporation of lysine into crosslinks in elastin.
Amino Acids↗
Determination of desmosine, isodesmosine, and other amino acids by liquid chromatography with electrochemical detection following precolumn derivatization with naphthalenedialdehyde/cyanide.
Naphthalenedialdehyde (NDA) in the presence of cyanide (CN) reacts with primary amines to produce fluorescent cyano[f]benzoisoindole (CBI) derivatives. These derivatives have been shown to be substantially more stable than the corresponding o-phthalaldehyde derivatives. However, one drawback of this method is that compounds derivatized at more than one site exhibit quenching, precluding the use of fluorescence detection. The CBI derivatives have been found to be electroactive and are oxidized at a modest oxidation potential (+750 mV). Electrochemical detection is especially useful for the analysis of compounds containing more than one primary amine site because the response is not attenuated as it is in fluorescence detection. Desmosine and isodesmosine were of particular interest because of their importance in elastic fiber and the lack of highly sensitive HPLC methods for the determination of these compounds. Both of these compounds react with NDA/CN to produce electrochemically active derivatives. The combination of derivatization with NDA/CN and electrochemical detection was found to be linear over three orders of magnitude. Detection limits for CBI-lysine and CBI-desmosine were 100 fmol at a S/N of 2. Amino acids in elastin were quantitated using this method. The results correlate well with what has been reported previously in the literature. A significant advantage of the use of liquid chromatography with electrochemical detection with precolumn derivatization with NDA/CN for the analysis of desmosine and isodesmosine is that they can be separated and quantitated individually using this method. In addition, the unique voltammetry of multiderivatized CBI-amino acids can be used to verify peak purity.
Aldehydes↗
Rapid method for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine and the amino sugars in proteins and tissues.
A rapid and sensitive chromatographic method is described for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine, glucosamine and galactosamine at picomole levels in protein and tissue hydrolysates. This method uses either an automated amino acid analyser with a 17.5 X 0.28 cm microcolumn packed with 6.0 +/- 0.5 micron spherical resin, thermostated at 52 degrees C, one buffer system (0.21 M sodium citrate, pH 5.125) and 3-nitrotyrosine as the internal standard, or conventional instruments using the same system but with larger diameter columns and resins (11.0 +/- 1.0 micron). This method should be especially valuable for determining collagen and elastin in tissue hydrolysates from the amounts of 5-hydroxylysine, and desmosine or isodesmosine present, respectively, and for studying protein hydroxylation, glycosylation, cross-linking formation, and the turnover rates of collagen and elastin in normal and diseased tissues.
Amino Acids↗
MEKC of desmosine and isodesmosine in urine of chronic destructive lung disease patients.
Degradation of extracellular matrix components is central to many pathological features of chronic destructive lung disorders. Desmosine and isodesmosine are elastin-derived cross-linked amino acids whose urine levels are considered representative of elastin breakdown. The aim of this study was to apply a novel methodology, based on high-performance capillary electrophoresis, to the quantification of desmosine and isodesmosine in 11 patients with stable chronic obstructive pulmonary disease (COPD), 10 with an exacerbation of COPD, nine with alpha1-antitrypsin deficiency, 13 with bronchiectasis, and 11 adults with cystic fibrosis, in comparison to 24 controls. It was found that, in patients with stable COPD, urinary desmosine levels were higher than in controls (p=0.03), but lower than in COPD subjects with an exacerbation (p< or =0.05). The highest desmosine levels were found in subjects with alpha1-antitrypsin deficiency, bronchiectasis and cystic fibrosis (p<0.001 versus stable COPD). In a short-term longitudinal study, five stable COPD patients showed a constant rate of desmosine excretion (mean coefficient of variation <8% over three consecutive days). In conclusion, the present method is simple and suitable for the determination of elastin-derived cross-linked amino acid excretion in urine, giving results similar to those obtained using other separation methods. In addition, evidence is presented that urinary desmosine excretion is increased in conditions characterized by airway inflammation, such as exacerbations of chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. Results obtained in subjects with alphal-antitrypsin deficiency suggest that this method might be used to evaluate the putative efficacy of replacement therapy.
Adult↗
Comparison of ELISA and HPLC for the determination of desmosine or isodesmosine in aortic tissue elastin.
We developed a rapid and simple method for estimating tissue elastin content by measuring desmosine (D) in tissue hydrolysates by competitive ELISA. We compared the ELISA previously reported HPLC methods. When D or isodesmosine (ID) in hydrolysate of the same elastin preparation were measured by the two different methods, a good linear relationship was obtained (r = 0.854 for human aorta or r = 0.938 for rabbit aorta, respectively). The ELISA method can detect as little as 6 pmol/ml and it may be useful in monitoring elastin metabolism in patients with various connective tissue diseases.
Animals↗
High-performance liquid chromatographic determination of desmosine and isodesmosine in tissues and its application to studies of alteration of elastin induced by atherosclerosis.
A rapid, sensitive high-performance liquid chromatographic method has been developed for the determination of desmosine (DES) and isodesmosine (IDE), the specific cross-linking amino acids of elastin, in the tissue hydrolysates of rats. DES and IDE in the hydrolysate samples were separated on a C18 column using 0.1 M phosphate buffer-acetonitrile (2.8:1) containing 20 mM sodium dodecyl sulphate (final pH 4.5) followed by detection at 270 nm. The recoveries of the added standards of DES and IDE from the aorta hydrolysate samples were 99.6 +/- 2.7% and 98.4 +/- 1.8%, respectively (n = 10). At DES and IDE concentrations of 2 micrograms/ml, within- and between-run precisions were 1.11-1.85% and 0.55-1.24%, respectively. The detection limits of DES and IDE were 0.1 microgram/ml with a 50-microliter injection at a signal-to-noise ratio of 3. The method was successfully applied to a study of the alteration of DES and IDE contents (i.e. elstin contents) in the tissues of rats treated with beta-aminopropionitrile and an atherogenic diet. A negative correlation between the contents of these amino acids and of cholesterol was noted in the atherosclerotic aorta.
Amino Acids↗
High-performance liquid chromatographic determination of desmosine and isodesmosine after phenylisothiocyanate derivatization.
A new sensitive and selective high-performance liquid chromatographic method for the analysis of desmosine and isodesmosine in human and rat tissues is described. This method requires a purification step with column chromatography, followed by precolumn derivatization phenylisothiocyanate. The reaction products are then separated by isocratic chromatography on a C18 column and quantitated by ultraviolet detection at 254 nm. The recovery of standards of both compounds added to tissue samples and analysed by this method is usually greater than 90%, and the absolute detection limit is 0.5 ng for both compounds. The method is sensitive enough to measure both substances in tissue fragments of 30 mg of wet mass, which means that it can be used to study elastin in small human biopsies.
Animals↗
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.
Chromatography, Liquid↗
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.
Animals↗
Micellar electrokinetic chromatography for the determination of urinary desmosine and isodesmosine in patients affected by chronic obstructive pulmonary disease.
The presence in urine of desmosine (DES) and isodesmosine (IDES), two crosslinked amino acids unique to the elastic fiber network, can be used as a specific indicator of degradation of mature elastin. Compared to methodologies so far available, the capillary electrophoretic technique reported here seems to be suitable and convenient for determining desmosines in urine of patients affected by chronic obstructive pulmonary disease (COPD). By using 35 mM sodium tetraborate pH 9.3 containing 65 mM SDS as the background electrolyte, the peaks of DES and IDES could be detected in hydrolyzed urine samples from controls and patients. Owing to the simultaneous determination of endogenous urinary creatinine used as appropriate internal standard, the amount of these amino acids could be accurately quantified. The results obtained were of the same order of magnitude as the data already reported in the literature for COPD patients. Thus micellar electrokinetic chromatography (MEKC) may be considered as a reliable technique for studying the turnover of the elastic fiber in clinical conditions.
Biomarkers↗
Desmosine and isodesmosine contents and elastase activity in normal and cirrhotic rat liver.
The contents of desmosine and isodesmosine, the cross-linking amino acids of elastin, were increased 4-fold in rat liver with carbon tetrachloride-induced cirrhosis, which suggests that insoluble elastin accumulates in cirrhosis. Elastase activity in the cirrhotic liver, as determined with 3-carboxypropionyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide, was 17% less than in the normal liver; no change was found when Congo Red-elastin was used as a substrate.
Amino Acids↗
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.
Adult↗
A large scale procedure for purification of desmosine and isodesmosine.
A rapid large scale procedure was devised for the purification of desmosine and isodesmosine from ligamentum nuchae elastin. The method makes use of the hydrophilic nature of the desmosines which preferentially absorbs to cellulose fibers in mixtures of organic solvents. Resolution of the isomers was achieved on a polystyrene resin column.
Amino Acids↗
Desmosine and isodesmosine as cross-links in the hinge-ligament protein of bivalves. 3,3'-Methylenebistyrosine as an artefact.
Desmosine and isodesmosine were detected in an invertebrate molluscan species, i.e. in an insoluble protein in the hinge ligament of a bivalve species, Sakhalin surf clam (Pseudocardium sachalinensis, in family Mactridae). The protein is rich in glycine and methionine S-oxide but devoid of hydroxyproline and hydroxylysine. 3,3'-Methylenebistyrosine was also detected in the HCl hydrolysate of the hinge-ligament protein, but it was found to be an artefact produced from tyrosine and formaldehyde derived from methionine S-oxide during the HCl hydrolysis of the protein.
Amino Acids↗