desmosine and Body-Weight

Elastin is one of the major extracellular matrix proteins associated with connective tissue. Its degradation leads to the liberation of the unique amino acids desmosine and isodesmosine. These have shown utility as biomarkers of elastin breakdown for disease progression, patient stratification, and drug efficacy. So far, the quantitation of desmosines in plasma is hampered by complex sample preparation. Here we demonstrate an improved and simplified procedure for detecting both free and total desmosines. The method is based on spiking with a deuterium-labeled desmosine standard, ethanol precipitation, propionylation, high-performance liquid chromatography (HPLC) separation, and selected reaction monitoring (SRM) mass spectrometry. The performance of the assay is illustrated by comparing the levels of free and total desmosines in normal healthy plasma and those from patients diagnosed with chronic obstructive pulmonary disease (COPD). A conserved ratio of 1:3 for free to total desmosine was found. The determination of free desmosine has higher accuracy than that of total desmosine; therefore, it is the method of choice when plasma volume is limiting. Finally, we show that the plasma desmosine concentration correlates with age and body mass index.

Topics: Adult; Age Factors; Aged; Biomarkers; Body Mass Index; Body Weight; Case-Control Studies; Chemical Precipitation; Chromatography, High Pressure Liquid; Desmosine; Deuterium; Female; Humans; Isodesmosine; Male; Mass Spectrometry; Middle Aged; Pulmonary Disease, Chronic Obstructive; Reference Values; Reproducibility of Results; Russia; Smoking; United States

The number of men with type II diabetes-associated erectile dysfunction (ED) continues to grow rapidly; however, the majority of basic science studies has examined mechanisms of ED in animal models of type I diabetes. In this study, we first establish an in vivo mouse model of type II diabetic ED using the leptin receptor mutated db/db and wild-type control BKS mouse. Furthermore, we hypothesized that dual mechanistic impairments contribute to the impaired erectile function in the type II diabetic mouse, altered vasoreactivity, and venoocclusive disorder. In vivo erectile function was measured as intracavernosal pressure (ICP) normalized to mean arterial pressure (MAP) following electrical stimulation of the cavernosal nerve. Venoocclusion was assessed by the maintenance of elevated in vivo ICP following intracorporal saline infusion. Vasoreactivity of isolated cavernosum in response to contractile and dilatory stimulation was examined in vitro by myography. Collagen and elastin content were evaluated by quantification of hydroxyproline and desmosine, respectively, as well as by quantitative PCR and histological analysis of isolated cavernosum. Erectile function was significantly decreased in db/db vs. BKS mice in a manner consistent with impairments in venoocclusive ability and decreased inflow. Heightened vasoconstriction and attenuated dilation in cavernosum of db/db vs. BKS mice suggest an overall lowered relaxation ability and thus impaired filling of the cavernosal spaces. A decrease in desmosine and hydroxyproline as well as lowered mRNA levels for tropoelastin, fibrillin-1, and alpha1(I) collagen were detected. These vasoreactive and sinusoidal matrix alterations may alter tissue compliance dispensability, preventing the normal expansion necessary for erection.

Topics: Acetylcholine; Animals; Blood Glucose; Blood Pressure; Body Weight; Collagen; Desmosine; Diabetes Mellitus, Type 2; Disease Models, Animal; Elastin; Electric Stimulation; Erectile Dysfunction; Extracellular Matrix; Hydroxyproline; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Myography; Organ Size; Penis; Phenylephrine; Polymerase Chain Reaction; Receptors, Leptin; Regional Blood Flow; RNA, Messenger; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Veins

In Marfan syndrome, mutations of the fibrillin gene (FBN1) lead to aneurysm of the thoracic aorta, making the aortic wall more susceptible to dissection, but the precise sequence of events underlying aneurysm formation is unknown. We used a rodent model of Marfan syndrome, the mgR/mgR mouse (with mgR: hypomorphic FBN1 mutation), which underexpresses FBN1, to distinguish between a defect in the early formation of elastic fibers and the later disruption of elastic fibers. The content of desmosine plus isodesmosine was used as an index of early elastogenesis; disruption of elastic fibers was analyzed by histomorphometry. Because disruption of the medial elastic fibers may produce aortic stiffening, so amplifying the aneurysmal process, we measured thoracoabdominal pulse wave velocity as an indicator of aortic wall stiffness. Both mgR/mgR and wild-type (C57BL/6J-129SV) strains were normotensive, and wall stress was not significantly modified because the increase in internal diameter (0.80+/-0.06 vs 0.63+/-0.03 mm in wild type, P<0.05) was accompanied by increased medial cross-sectional area. The aortic wall stiffened (4-fold increase in the elastic modulus-to-wall stress ratio). Desmosine content was not modified (mgR/mgR 432+/-31 vs wild type 492+/-42 microg/mg wet weight, P>0.05). Elastic fibers showed severe fragmentation: the percentage of the media occupied by elastic fibers was 18+/-3% in mgR/mgR mice vs 30+/-1% in wild-type mice, with the number of elastic segments being 1.9+/-0.2 vs 1.4+/-0.1x10(-6)/mm(2) in the wild type (both P<0.05). In conclusion, underexpression of FBN1 in mice leads to severe elastic network fragmentation but no change in cross-linking, together with aortic dilatation. This result suggests that fragmentation of the medial elastic network and not a defect in early elastogenesis is 1 of the determinants of aortic dilatation in Marfan syndrome.

Topics: Animals; Aorta; Aorta, Thoracic; Blood Pressure; Body Weight; Desmosine; Dilatation, Pathologic; Elastic Tissue; Elasticity; Female; Heart Rate; Isodesmosine; Male; Marfan Syndrome; Mice; Mice, Transgenic

We compared the ontogeny of collagen (hydroxyproline), elastin (desmosine), soluble protein, and DNA in the lungs of rate and humans during gestation and postnatal life. In humans, lung weight/body weight ratios declined faster during gestation than postnatally, whereas in rats lung weight/body weight ratio declined little during gestation and then suddenly on the first day of life. Lung weight/body weight ratios may be lower than expected around term in humans, and prediction data are given to assess human pulmonary hypoplasia. Rats and humans differed in water content of their lungs, with rats showing a sharper decline during gestation. In the human lung, collagen and elastin made their appearance at an early stage of gestation; elastin. In particular, increased rapidly during gestation, suggesting a role in intrauterine alveolar formation. In the rat, elastin accumulation is primarily a postnatal event, as is alveolar formation. Hydroxyproline concentrations increased with conceptual age and continued to increase rapidly postnatally between 4 and 7 weeks in the rat, but slowed in the human after 60 weeks of conceptual age. Desmosine concentrations level off at the end of the study period in rats, while these are still increasing, although slowly, in humans. Overall lung growth, as assessed by weight, was linear in humans, but phases of lung growth were apparent in the rat, including one of minimal growth in the immediate postnatal period.

Topics: Animals; Body Weight; Collagen; Desmosine; Female; Gestational Age; Humans; Hydroxyproline; Infant, Newborn; Lung; Organ Size; Pregnancy; Rats; Rats, Sprague-Dawley

Since Vitamin C (ascorbate, AH2) is an important airway antioxidant and is an essential component of tissue repair, and since acute (4 hr) O3 toxicity is enhanced by AH2 deficiency, we hypothesized that longer-term O3 effects might also be increased. Female Hartley guinea pigs (260-330 g) were fed either an AH2-sufficient or an AH2-deficient diet 1 week prior to exposure, and were maintained on their respective diets during 1 week of continuous exposure to O3 (0, 0.2, 0.4, and 0.8 ppm, 23 hr/day), and during 1 week postexposure recovery in clean air. The AH2-deficient diet caused lung AH2 to drop to about 30% of control in 1 week, and to below 10% by the end of exposure and recovery. Body weight gains during exposure were decreased in the 0.8 ppm O3 group, while the AH2 deficiency began to affect body weights only during recovery. O3 caused a concentration-dependent decrease in total lung capacity, vital capacity, carbon monoxide diffusing capacity, nitrogen washout, and static compliance, while increasing forced expiratory flow rates and residual or end-expiratory volume (suggestive of pulmonary gas-trapping). The lung/body weight ratio and fixed lung displacement volume were also increased in O3-exposed animals. Lung pathology consisted of mononuclear cell and neutrophil infiltration, airway as well as alveolar epithelial cell hyperplasia, and general decrease in epithelial cell cytoplasm. Thickening of the interstitium and an apparent increase in collagen staining were seen at the terminal bronchiolar regions. Some of these effects were marginally exacerbated in AH2-deficient guinea pigs. One week postexposure to air reversed all O3-induced abnormalities, irrespective of AH2 deficiency. Whole lung hydroxyproline and desmosine were not changed at any time by either O3 or AH2 deficiency. Measurement of lung prolyl hydroxylase activity suggested that AH2 deficiency as well as O3 exposure may have increased the tissue levels of this enzyme. The lack of a significant increase in toxicity with the longer-term exposure scenario suggests that AH2 has minimal influence on other compensatory mechanisms developed over time.

Topics: Animals; Ascorbic Acid; Ascorbic Acid Deficiency; Body Weight; Collagen; Desmosine; Elastin; Female; Guinea Pigs; Hydroxyproline; Lung Diseases; Lung Volume Measurements; Organ Size; Ozone; Procollagen-Proline Dioxygenase; Respiratory Function Tests; Tissue Fixation

To determine the role of calmodulin in postnatal lung growth and development, 4-week-old rats were injected intraperitoneally on consecutive days with trifluoperazine (TFP), a potent and specific calmodulin antagonist, for a period fo 3 weeks and studied in comparison with normal controls and undernourished weight-matched animals. TFP treatment resulted in stunting of lung growth such that observed normal increments in morphometrically determined total number of alveoli and alveolar surface area and in biochemically determined DNA, elastin, and collagen contents of the lungs were diminished in comparison with age-matched normal controls. However, the TFP treatment also resulted in reduced daily food intake and body weight gain. In the TFP group, lung weight and lung volume were also reduced compared with the weight-matched control group. This resulted in reduced alveolar surface area, total number of alveoli, DNA, collagen, and elastin in the TFP group compared with values in the weight-matched controls. Thus the TFP-induced lung changes were not due to inanition and/or reduced somatic growth. The TFP treatment resulted in reduced activities of calmodulin and cyclic adenosine monophosphate (cAMP)-phosphodiesterase in the lungs of the animals, independent of their nutritional status. Based on these findings, we suggest that calmodulin may be an important regulatory component of postnatal lung growth and development.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Animals; Body Weight; Calmodulin; Desmosine; DNA; Hydroxyproline; Lung; Male; Organ Size; Rats; Rats, Inbred Strains; RNA; Trifluoperazine

The ontogenesis of elastin and collagen accumulation and growth of the lung were studied in Wistar rats from day 18 of gestation until day 30 postnatally. Dexamethasone phosphate 0.1 mg or normal saline solution every 8 h for three doses was injected into pregnant rats on day 17. The effects of treatment, age, and sex on lung wet weight, lung dry weight, body weight, DNA, protein and desmosine (estimated by radioimmunoassay), and hydroxyproline were determined in the offspring. Dexamethasone inhibited lung growth and, to a lesser extent, body weight gain. While lung wet weight, lung dry weight, and body weight remained significantly reduced until postnatal day 15, the lung weight/body weight ratio was depressed only until postnatal day 5. The lung dry weight/lung wet weight ratio was decreased on day 20 of gestation and at birth. DNA concentration remained slightly but significantly reduced throughout the study period. Desmosine but not hydroxyproline concentration was lower after dexamethasone treatment during the period of rapid postnatal desmosine accumulation (day 10 p less than 0.05, day 15 p less than 0.01, day 20 p = 0.06).

Topics: Animals; Body Weight; Collagen; Desmosine; Dexamethasone; Elastin; Female; Fetus; Hydroxyproline; Lung; Male; Organ Size; Pregnancy; Rats; Rats, Inbred Strains

Emphysema is produced by severe food restriction in rats and is postulated to result from depletion of lung connective tissue. We studied (1) whether total dietary protein depletion worsens nutritional emphysema, and (2) whether the reduced content of lung connective tissue in nutritional emphysema results from lack of accumulation caused by impaired lung growth or by a net loss from the lung. Lewis rats weighing 200 g were restricted to one third food intake with or without protein for 6 wk. Lungs were assessed by morphometry, pressure-volume (P-V) measurements, and content of collagen and elastin. Emphysema was found by morphometry (but not by P-V measurements) in food-restricted rats, and contrary to expectation, emphysema was less severe in those depleted of protein. Collagen and elastin content were reduced in emphysematous lungs; however, the levels were not below those found prior to nutritional intervention, suggesting that lack of growth, not depletion, accounts for the reduced content.

Topics: Animals; Biomechanical Phenomena; Body Weight; Desmosine; Hydroxyproline; Lung; Lung Volume Measurements; Male; Organ Size; Protein Deficiency; Pulmonary Alveoli; Pulmonary Emphysema; Rats; Rats, Inbred Lew

The effects of ascorbic acid supplementation on the pulmonary toxicity induced by bleomycin were examined. Swiss-Webster mice were fed an ascorbate-free diet supplemented with ascorbic acid at 0%, 0.2%, or 1.0% of the diet for 2 weeks. Bleomycin (0.15 units) was instilled intratracheally and the mice were killed 1 week later. Bleomycin caused pulmonary inflammation and edema as noted by the increases in lung wet weight and lung wet-weight-to-dry-weight ratios. The activity of prolyl hydroxylase was increased 1.4-fold to 1.6-fold in response to bleomycin, but only minor increases were observed in the collagen and elastin content of the lung. Prior dietary ascorbic acid supplementation did not reverse the effects induced by bleomycin. Interestingly, each dietary level of supplemental ascorbic acid resulted in a slight increase in the elastin and collagen content of the lung in comparison with lungs from mice consuming no ascorbic acid in their diet. The data suggest that high levels of ascorbic acid supplementation may aggravate the response to bleomycin.

Topics: Animals; Ascorbic Acid; Bleomycin; Body Weight; Collagen; Connective Tissue; Desmosine; Diet; Drug Interactions; Elastin; Hydroxyproline; Lung; Mice; Organ Size; Procollagen-Proline Dioxygenase