Plasma Semicarbazide-sensitive Amine Oxidase Research Articles

Simulated microgravity alters the expression of plasma SSAO and its enzymatic activity in healthy rats and increases the mortality in high-fat diet/streptozotocin-induced diabetes

Semicarbazide-sensitive amine oxidase (SSAO) activity is known to be associated with certain pathologies including diabetes. In this study, SSAO expression and enzymatic activity in blood plasma of healthy rats were significantly upregulated under simulated microgravity (SMG) condition. Significant mortality was observed in SMG group of diabetic rats. Results indicate that microgravity might increase the risks of SSAO-associated alterations.

Plasma semicarbazide-sensitive amine oxidase activity in type 1 diabetes is related to vascular and renal function but not to glycaemia.

Associations of semicarbazide-sensitive amine oxidase (SSAO) activity with renal and vascular function, oxidative stress, glycaemia and diabetes complications were determined. Plasma SSAO activity in 94 type 1 diabetes (T1DM) patients, including 34 with microvascular complications T1DM CX[+], and in 96 healthy subjects (CON) was measured by production of benzaldehyde using high-performance liquid chromatography (HPLC). SSAO activity (mean ± SD) was greater in T1DM than in CON (1049 ± 294 vs 749 ± 204 mU/L; p < 0.00001) and was higher in T1DM CX[+] vs complication-free DM subjects (1148 ± 313 mU/L vs 982 ± 269 mU/L; p = 0.01). In T1DM, SSAO activity correlated with renal dysfunction [estimated glomerular filtration rate (eGFR): r = -0.44; p = 0.0001; cystatin C: r = 0.47; p = 0.0001] and markers of inflammation [soluble vascular cell adhesion molecule-1 (sVCAM-1): r = 0.41, p = 0.0001; soluble intercellular adhesion molecule-1 (sICAM-1): r = 0.33, p = 0.002] and was inversely related to small artery elasticity (SAE) (r = -0.23, p = 0.03). In CON, SSAO activity correlated with HbA1c (r = 0.26; p = 0.02). In T1DM, SSAO activity correlates with renal dysfunction, but not with glycaemia, and may promote vascular inflammation and be a therapeutic target.

Activity of semicarbazide-sensitive amine oxidase in guinea-pig tissues is not affected by metformin

Background Semicarbazide-sensitive amine oxidase (SSAO) is an enzyme that performs oxidative deamination of primary amines to aldehyde, ammonia and hydrogen peroxide. It appears that these products may have important signalling function in the regulation of cell development and glucose homeostasis. Much attention has been given to raised plasma SSAO activity in diabetic patients, both types 1 and 2. We were interested whether the peroral antidiabetic drug metformin affects SSAO activity in vitro. In this study we used metformin because it is usually the first-line medication used for treatment of type 2 diabetes.

Further Studies on the Metabolism of Methylamine by Semicarbazide-sensitive Amine Oxidase Activities in Human Plasma, Umbilical Artery and Rat Aorta

An ion exchange radiochemical assay has been developed to study the deamination of [14C]methylamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0.1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide-sensitive amino oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 microM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol (mg prot.)-1 h-1, and in plasma of 48 nmol (mL serum)-1 h-1. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 microM (plasma), 222 microM (umbilical artery), and Vmax values of 28 nmol (mL serum)-1 h-1 (plasma) and 377 nmol (mg prot.)-1 h-1 (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 microM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 microM) and [14C]MA (800 microM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of bovine plasma semicarbazide-sensitive amine oxidase by caffeine

Semicarbazide-sensitive amine oxidase (SSAO) is a copper-containing enzyme that catalyzes the oxidative deamination of endogenous and exogenous primary amines. SSAO exists in mammals both as a plasma-soluble and as a membrane-bound form, and its active site is able to come into contact with numerous xenobiotic, amine-containing compounds. The kinetic studies performed in this work showed that caffeine inhibition of bovine serum amine oxidase was noncompetitive when benzylamine was used as substrate and mixed when the substrate used was methylamine. Since caffeine contains an imidazole ring, it cannot be excluded that it might bind to an inhibitory imidazoline-binding site on SSAO.

Modulation of Endothelium-Dependent Relaxation of Equine Digital Arteries By Insulin and Hypoxia

Modulation of Endothelium-Dependent Relaxation of Equine Digital Arteries By Insulin and Hypoxia

P4-299: Neuron-binding autoantibodies in human sera enhance Abeta42 accumulation in adult mouse brain neurons

of Alzheimer’s disease (AD), and it is characterized by the deposition of beta-amyloid (A ) in cerebral cortical and meningeal blood vessels (Kawai M. et al., 1993), inducing degeneration of vascular cells. Semicarbazide-sensitive amine oxidase (SSAO) [E.C. 1.4.3.6] is present in vascular cells and in plasma. It metabolizes primary amines (Lyles GA., 1996) generating hydrogen peroxide (H2O2), ammonia (NH3) and the corresponding aldehyde, that contribute to the oxidative stress, advanced glycation end-product generation (GubisneHaberle D. et al., 2004), and beta amyloid aggregation (Chen K. et al., 2006). Furthermore in endothelial cells, SSAO is induced under inflammatory conditions (Smith D.J. et. al., 1998). We have reported that SSAO is overexpressed in cerebrovascular tissue of patients with CAA-AD, and that it colocalizes with beta-amyloid deposits (Ferrer I. et al., 2002). This over-expression correlates with high SSAO activity in plasma of severe AD patients (del Mar Hernandez M. et al., 2005). We have also described that plasma SSAO is able to induce apoptosis in vascular cells (Hernandez M. et.al., 2006). The aim of this work is to demonstrate whether A is able to induce SSAO overexpression in HUVEC cells as vascular cell type. Methods: Because of the SSAO/ VAP-1 expression fenotype is lost in cultured cells, HUVEC (human umbilical vein endothelial cells) cells, were stably transfected with vector pcDNA 3.1 containing hVAP-1/SSAO. Cells were treated with A 1-40 Dutch type (mutation E22Q) and/or Methylamine as SSAO substrate. Cell viability, SSAO activity and its expression were determined using specific antibodies against SSAO. Results: Herein we report that A 1-40 E22Q induces the SSAO activity and expression in HUVEC cells. This increasing activity promotes oxidative stress that enhances the toxicity generated by A alone. This toxicity is reverted by specific SSAO inhibitors, confirming SSAO as the responsible of such effect. Conclusions: These results allow us to postulate that deposits of A in cerebrovascular tissue, induce SSAO expression that may contribute to the vascular damage associated to CAA-AD.

Synergistic interaction between semicarbazide-sensitive amine oxidase and angiotensin-converting enzyme in diabetes: functional analysis by gene ontology

Plasma semicarbazide-sensitive amine oxidase (SSAO) and angiotensin-converting enzyme (ACE) were studied for their correlation with diabetes (DM) complication. The effect of interaction between SSAO and ACE in DM complication is of interest. Studying the functional change due to interaction between SSAO and ACE is difficult. In this work, the author used a new gene ontology technology to predict the functional change resulting from the interaction between SSAO and ACE. According to this study, there is a synergetic effect resulting from the interaction between SSAP and ACE. This can imply that co-expression of SSAP–ACE leads to more severe complication of DM. However, the author can also demonstrate that some molecular functions such as proteasome activator activity of SSAO and hydrolase activity, metallopeptidase activity, and zinc ion binding of ACE are suppressed after co-expression. These results provide good information for further study in diabetes medicine. However, further experimental works are required to support this in silico research.

Sodium Bicarbonate Enhances Membrane-bound and Soluble Human Semicarbazide-sensitive Amine Oxidase Activity In Vitro

Semicarbazide-sensitive amine oxidase (SSAO) is a multifunctional enzyme with different biological roles that depend on the tissue where it is expressed. Because SSAO activity is altered in several pathological conditions, we were interested in studying the possible regulation of the human enzyme activity. It has been previously reported that SSAO activity is increased in the presence of Dulbecco's modified Eagle medium (DMEM) in vitro. The aim of the present work was to investigate the effects of the different constituents of DMEM on human SSAO activity. We found that sodium bicarbonate was the only component able to mimic the enhancement of both human aorta and plasma SSAO activity in vitro, suggesting a possible physiological role of bicarbonate as an intrinsic modulator of the human enzyme. Failure to take this activating effect into account could also result in inaccuracies in the reported tissue activities of this enzyme.

Soluble Semicarbazide Sensitive Amine Oxidase (SSAO) catalysis induces apoptosis in vascular smooth muscle cells

Semicarbazide sensitive amine oxidase (SSAO) metabolizes oxidative deamination of primary aromatic and aliphatic amines. It is selectively expressed in vascular cells of blood vessels, but it is also circulating in blood plasma. SSAO activity in plasma is increased in some diseases associated with vascular complications and its catalytic products may cause tissue damage. We examined the effect of the oxidation of the SSAO substrate, methylamine, on cultured smooth muscle cells. Cell incubation with methylamine plus soluble SSAO, contained in bovine serum, resulted toxic to rat aorta A7r5 and human aortic smooth muscle cells, as measured by MTT reduction. This effect was completely reverted by specific SSAO inhibitors, indicating that the toxicity was mediated by the end products generated. Moreover, SSAO-mediated deamination of methylamine induced apoptosis in A7r5 cells, detected by chromatin condensation, Caspase-3 activation, PARP cleavage and cytochrome c release to cytosol. Formaldehyde, rather than H 2O 2, resulted to be a strong apoptotic inducer to A7r5 cells. Taken together, the results suggest that increased plasma SSAO activity in pathological conditions, could contribute to apoptosis in smooth muscle cells, leading to vascular tissue damage.

Plasma semicarbazide‐sensitive amine oxidase is moderately decreased by pronounced exogenous hyperinsulinemia but is not associated with insulin sensitivity and body fat

Objective. Semicarbazide‐sensitive amine oxidase (SSAO) is widely expressed in adipose tissue, where it may contribute to stimulation of glucose transport via GLUT4 recruitment. We tested the relationships of soluble SSAO, as reflected by its plasma activity, with insulin sensitivity and indices of body fat, and determined whether insulin is involved in regulating plasma SSAO activity. Material and methods. In 24 non‐diabetic subjects, the relationships of plasma SSAO activity with insulin sensitivity (M‐value and free fatty acid (FFA) suppression during a 3‐h hyperinsulinemic (8.3 µU kg−1 s−1), euglycemic clamp), body mass index (BMI 25.5±3.1 kg m−2), waist‐hip ratio and fat mass were assessed. In 16 subjects, the effect of insulin infusion, administered at a rate of 8.3 µU kg−1 s−1 during 3 h, followed by 3‐h insulin infusion at a high rate of 41.7 µU kg−1 s−1 on plasma SSAO activity was determined. In the other 8 subjects, the response of plasma SSAO activity to 24‐h insulin infused at 8.3 µU kg−1 s−1 was assessed. Results. There were no relationships (all p >0.10) of plasma SSAO activity (215±60 mU L−1) with the M‐value or with any indices of body fat and FFA before and after insulin suppression. Plasma SSAO activity changed by −7.2 (95% CI, −14.5 to +0.2)% after 3 h (NS) and decreased by 10.1 (95% CI, 19.2 to 1)% after 6 h of insulin infusion (p <0.05). Plasma SSAO activity did not significantly change after 8 h (change 0.4 (95% CI, −15.3 to +16.2)%, NS) and after 24 h (change −8.8 (95% CI, −27.4 to +9.7)%, NS) of insulin infusion. Conclusions. It is unlikely that circulating SSAO is a clinically important marker of insulin sensitivity on glucose and fatty acid metabolism. The reduction in plasma SSAO activity in response to pronounced hyperinsulinemia suggests that insulin is involved in the regulation of the soluble form of this enzyme.

Glucose handling in streptozotocin-induced diabetic rats is improved by tyramine but not by the amine oxidase inhibitor semicarbazide

A soluble form of semicarbazide-sensitive amine oxidase (SSAO) circulating in plasma is known to increase in type 1 and 2 diabetes. This cuproenzyme generates hydrogen peroxide, ammonia, and aldehydes when oxidizing circulating biogenic or exogenous amines. Based on the angiotoxicity of these products, inhibition of SSAO has been proposed to prevent vascular complications of diabetes. However, substrates of SSAO and monoamine oxidase (MAO) have been recently evidenced to activate glucose utilisation in insulin-sensitive tissues and to exhibit antihyperglycemic actions. To determine whether amine oxidase blockade or activation could be beneficial for diabetes, we aimed at comparing the influence of prolonged treatments with semicarbazide (SSAO-inhibitor), pargyline (MAO-inhibitor), or tyramine (amine oxidase substrate) on amine oxidase activities and glycemic control in streptozotocin-induced diabetic rats. The increase in plasma SSAO was confirmed in diabetic rats, while MAO and SSAO were decreased in subcutaneous adipose tissue when compared with normoglycemic controls. Among the diabetic rats, only those receiving tyramine exhibited slightly decreased hyperglycemia and improved glucose tolerance. Adipocytes from untreated or treated diabetic rats shared similar sensitivity to insulin. However glucose uptake activation and lipolysis inhibition in response to amine oxidase substrates combined with vanadate were impaired in rats treated with amine oxidase inhibitors. Thus, amine oxidase inhibition does not improve metabolic control while prolonged administration of tyramine slightly improves glucose disposal. It is therefore concluded that amine oxidase activation by increased substrate supply elicits insulin-like actions that may be more beneficial in diabetes than SSAO inhibition formerly proposed to prevent vascular complications.

Human plasma semicarbazide sensitive amine oxidase (SSAO), β-amyloid protein and aging

Semicarbazide sensitive amine oxidase (SSAO) metabolizes oxidative deamination of primary aromatic and aliphatic amines. The final products of its catalysis, ammonia, hydrogen peroxide (H 2O 2) and the corresponding aldehyde, may contribute to diseases involving vascular degeneration. SSAO is selectively expressed in blood vessels in the brain, but is also present in blood plasma. We have previously reported that membrane-bound SSAO is overexpressed in the cerebrovascular tissue of Alzheimer's disease (AD) patients. The aim of the present work is to study whether the circulating SSAO is also altered in this neurodegenerative disease. SSAO activity was determined in plasma of control cases ( n = 23) and patients suffering sporadic Alzheimer dementia, distributed according to the Global Deterioration Scale (GDS): mild ( n = 33), moderate ( n = 14), moderate–severe ( n = 15) and severe dementia ( n = 19). Results show a clear increase of plasma SSAO activity ( p < 0.001) in moderate–severe and severe AD patients, with patient age being an independent correlative factor. However, plasma SSAO activity was not altered in AD patients with mild or moderate dementia compared to controls. β-Amyloid (Aβ) (40–42) immunoreactivity in plasma samples was also determined, and no correlation was observed between Aβ 40–42 levels and the severity of the dementia or the plasma SSAO activity. Our results suggest that an increase in circulating SSAO activity could contribute to oxidative stress and vascular damage in advanced Alzheimer's disease.

Association between plasma activities of semicarbazide-sensitive amine oxidase and angiotensin-converting enzyme in patients with type 1 diabetes mellitus

Plasma semicarbazide-sensitive amine oxidase (SSAO) is elevated in patients with type 1 and type 2 diabetes and has been implicated in the pathophysiology of diabetic late complications. The regulation of SSAO production remains unknown. We studied correlations between plasma SSAO activity and parameters associated with diabetic late complications. Plasma SSAO was measured in a well-characterised group of 287 patients with type 1 diabetes. Standard statistical methods were used to investigate correlations with clinical parameters and components of the renin-angiotensin system. Overall, plasma SSAO was elevated, at 693+/-196 mU/l (mean+/-SD; normal controls 352+/-102 mU/l). Plasma SSAO was higher in the group with late complications or hypertension, and in patients treated with ACE-inhibitors. In univariate analysis a significant positive correlation (p<0.001, r=0.27) was found between plasma SSAO and serum ACE activity in patients untreated with ACE inhibitors or angiotensin II receptor antagonists (n=221), but plasma SSAO did not differ by ACE I/D genotype. Plasma SSAO correlated positively with duration of diabetes, HbA(1)c and plasma renin, and negatively with plasma angiotensinogen and body mass index. A multiple regression analysis including these variables resulted in serum ACE activity (p<0.001), ACE genotype (negatively, p<0.001) and HbA(1)c (p=0.023) as explaining variables. Results suggest that a common factor is involved in the regulation of both plasma SSAO and serum ACE, which is different from the genetic determination of ACE activity.

0740 Plasma semicarbazide sensitive amine oxidase (ssao) is elevated in alzheimer dementia: correlation with the neurological disorder severity

0740 Plasma semicarbazide sensitive amine oxidase (ssao) is elevated in alzheimer dementia: correlation with the neurological disorder severity

Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography

Semicarbazide-sensitive amine oxidase (SSAO) is present in plasma, as well as in other tissues. Previous studies indicated that SSAO is of important physiological and pathophysiological functions. HPLC methods were developed for the assay of SSAO in plasma, and for the determination of plasma methylamine, an SSAO's endogenous substrate. Benzylamine was used as artificial substrate for the enzyme activity assay of SSAO. A 0.2-ml aliquot of plasma was incubated with benzylamine at 37 °C for 30 min. Benzaldehyde, the enzymatic reaction product, was derivatized with 2,4-dinitrophenylhydrazine (DNPH), and analyzed with HPLC and UV detection. SSAO enzyme activity is defined as benzaldehyde (nmol) formed per ml plasma per hour. Recoveries of benzaldehyde spiked to plasma were between 63.5 and 68.2% with relative standard deviation less than 3%. To determine methylamine in plasma, a 0.1-ml aliquot of plasma was deproteinized by trichloroacetic acid and centrifugation. The supernatant was derivatized with dansyl chloride and analyzed by HPLC with fluorescence detection. Recoveries of spiked methylamine at ppb (ng/ml) level were between 93.7 and 97.6% with relative standard deviation less than 2.5%.

Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography

Semicarbazide-sensitive amine oxidase (SSAO) is present in plasma, as well as in other tissues. Previous studies indicated that SSAO is of important physiological and pathophysiological functions. HPLC methods were developed for the assay of SSAO in plasma, and for the determination of plasma methylamine, an SSAO's endogenous substrate. Benzylamine was used as artificial substrate for the enzyme activity assay of SSAO. A 0.2-ml aliquot of plasma was incubated with benzylamine at 37 °C for 30 min. Benzaldehyde, the enzymatic reaction product, was derivatized with 2,4-dinitrophenylhydrazine (DNPH), and analyzed with HPLC and UV detection. SSAO enzyme activity is defined as benzaldehyde (nmol) formed per ml plasma per hour. Recoveries of benzaldehyde spiked to plasma were between 63.5 and 68.2% with relative standard deviation less than 3%. To determine methylamine in plasma, a 0.1-ml aliquot of plasma was deproteinized by trichloroacetic acid and centrifugation. The supernatant was derivatized with dansyl chloride and analyzed by HPLC with fluorescence detection. Recoveries of spiked methylamine at ppb (ng/ml) level were between 93.7 and 97.6% with relative standard deviation less than 2.5%.

Hypertensive chronic heart failure – value of plasma semicarbazide-sensitive amine oxidase as an useful tool for the diagnosis and follow-up of patients with left ventricular systolic dysfunction

OR-67 Key Words: Plasma Semicarbazide-Sensitive Amine Oxidase, Brain Natriuretic Peptide, Endothelin-1

Adipocytes release a soluble form of VAP-1/SSAO by a metalloprotease-dependent process and in a regulated manner.

Vascular adhesion protein-1 (VAP-1), which is identical to semicarbazide-sensitive amine oxidase (SSAO), is a dual-function membrane protein with adhesion properties and amine oxidase activity. A soluble form of VAP-1 is found in serum, where concentrations are enhanced in diabetes and obesity. In vitro, soluble VAP-1 enhances lymphocyte adhesion to endothelial cells, thus possibly participating in the enhanced lymphocyte adhesion capacity that is implicated in the cardiovascular complications associated with diabetes or obesity. In both, the tissue origin of the soluble VAP-1/SSAO is unknown. We examined whether adipose tissue, which has abundant expression of VAP-1/SSAO, is a source of soluble VAP-1. We detected VAP-1/SSAO in plasma of diabetic animals, with or without VAP-1 immunoprecipitation, and in culture medium from 3T3-L1 adipocytes and human adipose tissue explants. VAP-1 protein glycosylation was measured. Diabetic and obese animals have increased plasma SSAO activity associated with VAP-1 protein. We also found that 3T3-L1 adipocytes and human adipose tissue explants release a soluble form of VAP-1/SSAO, which derives from the membrane. The release of soluble VAP-1 was enhanced by exposure of murine and human adipocytes to TNF-alpha and blocked by batimastat, a metalloprotease inhibitor. Partial ablation of adipose tissue reduced plasma SSAO activity in normal and diabetic rats. Adipose cells are a source of soluble VAP-1/SSAO released by shedding of the membrane form. The release of SSAO is regulated by TNF-alpha and insulin. By releasing VAP-1/SSAO, adipose cells could contribute to the atherogenesis and vascular dysfunction associated with diabetes and obesity.

Overexpression of Semicarbazide-Sensitive Amine Oxidase in Smooth Muscle Cells Leads to an Abnormal Structure of the Aortic Elastic Laminas

Elevated semicarbazide-sensitive amine oxidase (SSAO) activity has been observed in several human conditions, eg, diabetes, and it has been speculated that SSAO contributes to the development of vasculopathies associated with this disease. To investigate in vivo consequences of elevated expression of SSAO in vascular tissues, we have developed a transgenic model for overexpression of human SSAO in mice. A smooth muscle-specific promoter, smooth muscle alpha-actin promoter 8 (SMP8) was used. Transgenic expression of human SSAO in tissues with a high content of smooth muscle cells was confirmed by Northern blot analysis. Enzymatic analysis of homogenates from transgenic tissues showed elevated levels of SSAO activity compared to non-transgenic littermates. Furthermore, when plasma SSAO activity was analyzed, much higher activity was detected compared to plasma from control mice, indicating that plasma SSAO may originate from smooth muscle cells. Histopathological evaluation of aorta and renal artery from transgenic mice revealed an abnormal structure of the elastin tissue. Instead of the regularly folded elastic laminae normally found in tunica media of sacrificed mice, the elastic laminae were straight and unfolded with irregularly arranged elastic fibers, forming tangled webs, between the intercalating elastic laminae. These alterations of the elastin structures suggest that overexpression of SSAO has led to a reduced elasticity of the arteries. Moreover, the mean femoral arterial pressure of the SMP8 SSAO transgenic mice was significantly lower in comparison to non-transgenic littermates. This suggests that the transgenic mice have a defect in their ability to regulate blood pressure.