Semicarbazide-sensitive Amine Oxidase Research Articles

Correlation among soluble receptors for advanced glycation end-products, soluble vascular adhesion protein-1/semicarbazide-sensitive amine oxidase (sVAP-1) and cardiometabolic risk markers in apparently healthy adolescents: a cross-sectional study

In non-diabetics, low levels of soluble receptor for advanced glycations end products (sRAGE) associate with an increased risk of development of diabetes, cardiovascular afflictions, or death. The majority of studies in non-diabetics report an inverse relationship between measures of obesity, cardiometabolic risk factors and sRAGE and/or endogenous secretory RAGE (esRAGE) levels. To elucidate whether this inconsistency is related to the metabolically healthy obese phenotype, or a different impact of the risk factors in presence and absence of obesity, we analyzed data from 2206 apparently healthy adolescents (51% girls) aged 15-to-19years. The association of sRAGE levels with soluble vascular adhesion protein-1/semicarbazide sensitive amine oxidase (sVAP-1/SSAO) was also investigated. Centrally obese, including metabolically healthy, adolescents present significantly lower sRAGE and esRAGE, but not sVAP-1, levels in comparison with their lean counterparts. An increasing number of cardiometabolic risk factors did not associate with significant changes in sRAGE, esRAGE or sVAP-1 levels either in lean or in obese subjects. In multivariate analyses, WHtR, hsCRP, markers of glucose homeostasis, renal function, adiponectin, and sVAP-1 associated significantly with sRAGE and esRAGE. SVAP-1 correlated significantly with glycemia, adiponectin, hsCRP, and sRAGE. Thus, in adolescents, a decline in sRAGE and esRAGE precedes the development of metabolic syndrome. When combined, standard and non-standard cardiometabolic risk factors explain only minor proportion in a variability of sRAGE and esRAGE (8%-11%); or sVAP-1 (12%-20%). Elucidation of pathogenetic mechanisms underlying early decline in sRAGE and esRAGE levels in obese adolescents and their clinical impact with regard to future cardiometabolic health requires further studies.

Methyl Jasmonate Elicitation Affects Expression of Genes Involved in Biosynthesis and Turnover of 2-Phenylethylamine in Maize Seedlings

Abstract The objective of the study was to assess the influence of methyl jasmonate (MJ) vapors on accumulation of 2-phenylethylamine (PEA), phenylacetic acid (PAA) and 2-phenylethanol (PE) in leaves and roots of maize (Zea mays L. subsp. mays, saccharata group, cv. Złota Karłowa) seedlings. Furthermore, we analyzed the expression patterns of eight genes (ADH1, ADH2, AO2, CAO, PDC1, PDC2, PTA and LOX, encoding alcohol dehydrogenase 1 and 2, primary amine oxidase, aldehyde oxidase 2, phenylalanine decarboxylase 1 and 2, phenylalanine (histidine) transaminase and lipoxygenase, respectively) involved in biosynthesis and turnover of PEA in maize tissues. In addition, the effect of MJ application on fresh biomass and growth of the tested seedlings was recorded. One-day MJ exposure increased the fresh weight of aerial parts and roots of Z. mays seedlings, whereas the opposite tendency occurred after 4-day of MJ treatment. One-day application of MJ resulted in an increase in the length of roots and its fluctuations in the aerial parts of maize plants, but extended exposure declined the growth of both parts of the seedlings. Methyl jasmonate elicitation caused various changes in the contents of PEA, PAA and PE in the maize seedlings. MJ treatments led to high upregulation of most genes, with the exception of three genes (i.e., ADH1, ADH2 and AO2) whose expression was downregulated after a 4-day exposure.

Triethylenetetramine modulates polyamine and energy metabolism and inhibits cancer cell proliferation.

Polyamine metabolism is an attractive anticancer drug target, since polyamines are absolutely required for cellular proliferation, and increased levels of polyamines and their biosynthetic enzyme ornithine decarboxylase (ODC) are associated with cancer. Triethylenetetramine (TETA) is a charge-deficient isosteric analogue of the polyamine spermidine (Spd) and a Cu(II)-chelating compound used for the treatment of Wilson's disease, and it has been implicated as a potential anticancer therapeutic drug. In the present study, we studied the effects of TETA in comparison with two other Cu(II)-chelators, D-penicillamine (PA) and tetrathiomolybdate (TTM), on polyamine metabolism in DU145 prostate carcinoma, MCF-7 breast carcinoma and JEG-3 choriocarcinoma cells. TETA induced antizyme, down-regulated ODC and inhibited [(14)C] Spd uptake. Moreover, it completely prevented α-difluoromethylornithine (DFMO)-induced increase in [(14)C] Spd uptake, and inhibited [(14)C] putrescine (Put) uptake and ODC activity invivo Seven-day treatment of DU145 cells with TETA caused growth cessation by reducing intracellular polyamine levels and suppressing the formation of hypusinated eukaryotic translation initiation factor 5A (eIF5A). TETA or its N-acetylated metabolites also inhibited spermine (Spm), diamine and semicarbazide-sensitive amine oxidases and decreased the level of intracellular reactive oxygen species. Moreover, TETA inhibited the utilization of Put as energy source via the tricarboxylic acid (TCA) cycle, as indicated by decreased production of (14)CO2 from [(14)C] Put. These results indicate that TETA attacks multiple proven anticancer drug targets not attributed to copper chelation, which warrants further studies to reveal its potential in cancer chemoprevention and cure.

HPLC-UV method for evaluation of inhibitors of plasma amine oxidase using derivatization of an aliphatic aldehyde product with TRIS.

Plasma amine oxidase (PAO), which is also designated as semicarbazide-sensitive amine oxidase (SSAO), copper-containing amine oxidase 3 (AOC3), or vascular adhesion protein-1 (VAP-1), catalyzes the oxidative deamination of primary amines to aldehydes using copper and a quinone as cofactors. Because it participates in the transmigration of inflammatory cells through the blood vessels into the tissue, PAO is attributed an important role in inflammatory diseases. Therefore, inhibitors of this enzyme could lead to new therapeutics for the treatment of inflammation-related conditions. Assays for the evaluation of PAO inhibitors usually measure the conversion of benzylamine to benzaldehyde by UVspectroscopy. We have developed a test system with the new substrate 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine, monitoring the formation of the enzyme product 6-(5-phenyl-2H-tetrazol-2-yl)hexanal by reversed phase HPLC with UV detection. Since this compound only eluted with poor peak shape due to hydrate formation in the aqueous mobile phase, it was derivatized with tris(hydroxymethyl)aminomethane (TRIS) under mild conditions to an oxazolidine prior HPLC analysis. The validation of the method revealed that the new substrate was bound with higher affinity to PAO and converted with higher velocity than the standard substrate benzylamine.

Inactivation of Semicarbazide-Sensitive Amine Oxidase Stabilizes the Established Atherosclerotic Lesions via Inducing the Phenotypic Switch of Smooth Muscle Cells.

Given that the elevated serum semicarbazide-sensitive amine oxidase (SSAO) activity is associated with the severity of carotid atherosclerosis in clinic, the current study aims to investigate whether SSAO inactivation by semicarbazide is beneficial for established atherosclerotic lesions in LDLr knockout mice on a high-fat/high- cholesterol Western-type diet or after dietary lipid lowering. Despite no impact on plasma total cholesterol levels, the infiltration of circulating monocytes into peripheral tissues, and the size of atherosclerotic lesions, abrogation of SSAO activity resulted in the stabilization of established lesions as evidenced by the increased collagen contents under both conditions. Moreover, SSAO inactivation decreased Ly6Chigh monocytosis and lesion macrophage contents in hypercholesterolemic mice, while no effect was observed in mice after normalization of hypercholesterolemia by dietary lipid lowering. Strikingly, abrogation of SSAO activity significantly increased not only the absolute numbers of smooth muscle cells (SMCs), but also the percent of SMCs with a synthetic phenotype in established lesions of mice regardless of plasma cholesterol levels. Overall, our data indicate that SSAO inactivation in vivo stabilizes the established plaques mainly via inducing the switch of SMCs from a contractile to a synthetic phenotype. Targeting SSAO activity thus may represent a potential treatment for patients with atherosclerosis.

Inactivation of semicarbazide-sensitive amine oxidase induces the phenotypic switch of smooth muscle cells and aggravates the development of atherosclerotic lesions

Background and aimsClinical studies have demonstrated that serum semicarbazide-sensitive amine oxidase (SSAO) activities positively correlate with the progression of atherosclerosis. The aim of the present study is to investigate the effect of SSAO inactivation on the development of atherosclerosis. MethodsFemale LDLr knockout (KO) mice were given the Western-type diet for 6 and 9 weeks to induce the formation of early and advanced lesions, and semicarbazide (SCZ, 0.125%) was added into the drinking water to inactivate SSAO in vivo. ResultsDespite no impact on plasma total cholesterol levels, abrogation of SSAO by SCZ not only resulted in the enlargement of both early (1.5-fold, p = 0.0043) and advanced (1.8-fold, p = 0.0013) atherosclerotic lesions, but also led to reduced/increased lesion contents of macrophages/smooth muscle cells (SMCs) (macrophage: ∼0.74-fold, p = 0.0002(early)/0.0016(advanced); SMC: ∼1.55-fold, p = 0.0003(early)/0.0001(advanced)), respectively. Moreover, SSAO inactivation inhibited the migration of circulating monocytes into peripheral tissues and reduced the amount of circulating Ly6Chigh monocytes (0.7-fold, p = 0.0001), which may account for the reduced macrophage content in lesions. In contrast, the increased number of SMCs in lesions of SCZ-treated mice is attributed to an augmented synthetic vascular SMC phenotype switch as evidenced by the increased proliferation of SMCs and accumulation of collagens in vivo. ConclusionSSAO inactivation by SCZ promotes the phenotypic switch of SMCs and the development of atherosclerosis. The enzymatic activity of SSAO may thus represent a potential target in the prevention and/or treatment of atherosclerosis.

Amine oxidases as important agents of pathological processes of rhabdomyolysis in rats.

In this study we have tested an idea on the important role of amine oxidases (semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase) as an additional source of oxidative/carbonyl stress under glycerol-induced rhabdomyolysis, since the enhanced formation of reactive oxygen species and reactive carbonyl species in a variety of tissues is linked to various diseases. In our experiments we used the sensitive fluorescent method devised for estimation of amine oxidases activity in the rat kidney and thymus as targeted organs under rhabdomyolysis. We have found in vivo the multiple rises in activity of semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase (2-4.5 times) in the corresponding cell fractions, whole cells or their lysates at the 3-6th day after glycerol injection. Aberrant antioxidant activities depended on rhabdomyolysis stage and had organ specificity. Additional treatment of animals with metal chelator ‘Unithiol’ adjusted only the activity of antioxidant enzymes but not amine oxidases in both organs. Furthermore the in vitro experiment showed that Fenton reaction (hydrogen peroxide in the presence of iron) products alone had no effect on semicarbazide-sensitive amine oxidase activity in rat liver cell fraction whereas supplementation with methylglyoxal resulted in its significant 2.5-fold enhancement. Combined action of the both agents had additive effect on semicarbazide-sensitive amine oxidase activity. We can assume that biogenic amine and polyamine catabolism by amine oxidases is upregulated by oxidative and carbonyl stress factors directly under rhabdomyolysis progression, and the increase in catabolic products concentration contributes to tissue damage in glycerol-induced acute renal failure and apoptosis stimulation in thymus.

Expression of the semicarbazide-sensitive amine oxidase in articular cartilage: its role in terminal differentiation of chondrocytes in rat and human

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the oxidation of primary amines into ammonia and reactive species (hydrogen peroxide, aldehydes). It is highly expressed in mammalian tissues, especially in vascular smooth muscle cells and adipocytes, where it plays a role in cell differentiation and glucose transport. The study aims at characterizing the expression and the activity of SSAO in rat and human articular cartilage of the knee, and to investigate its potential role in chondrocyte terminal differentiation. SSAO expression was examined by immunohistochemistry and western blot. Enzyme activity was measured using radiolabeled benzylamine as a substrate. Primary cell cultures of rat chondrocytes were treated for 21 days by a specific SSAO inhibitor, LJP 1586. Terminal chondrocyte differentiation markers were quantified by RT-qPCR. The basal and IL1β-stimulated glucose transport was monitored by the entrance of (3)[H]2-deoxyglucose in chondrocytes. SSAO was expressed in chondrocytes of rat and human articular cartilage. SSAO expression was significantly enhanced during the hypertrophic differentiation of chondrocytes characterized by an increase in MMP13 and in alkaline phosphatase (ALP) expressions. SSAO inhibition delayed the late stage of chondrocyte differentiation without cell survival alteration and diminished the basal and IL1β-stimulated glucose transport. Interestingly, SSAO activity was strongly increased in human osteoarthritic cartilage. SSAO was expressed as an active form in rat and human cartilage. The results suggest the involvement of SSAO in rat chondrocyte terminal differentiation via a modulation of the glucose transport. In man, the increased SSAO activity detected in osteoarthritic patients may trigger hypertrophy and cartilage degeneration.

Dietary Phenolic Compounds Interfere with the Fate of Hydrogen Peroxide in Human Adipose Tissue but Do Not Directly Inhibit Primary Amine Oxidase Activity.

Resveratrol has been reported to inhibit monoamine oxidases (MAO). Many substrates or inhibitors of neuronal MAO interact also with other amine oxidases (AO) in peripheral organs, such as semicarbazide-sensitive AO (SSAO), known as primary amine oxidase, absent in neurones, but abundant in adipocytes. We asked whether phenolic compounds (resveratrol, pterostilbene, quercetin, and caffeic acid) behave as MAO and SSAO inhibitors. AO activity was determined in human adipose tissue. Computational docking and glucose uptake assays were performed in 3D models of human AO proteins and in adipocytes, respectively. Phenolic compounds fully inhibited the fluorescent detection of H2O2 generated during MAO and SSAO activation by tyramine and benzylamine. They also quenched H2O2-induced fluorescence in absence of biological material and were unable to abolish the oxidation of radiolabelled tyramine and benzylamine. Thus, phenolic compounds hampered H2O2 detection but did not block AO activity. Only resveratrol and quercetin partially impaired MAO-dependent [14C]-tyramine oxidation and behaved as MAO inhibitors. Phenolic compounds counteracted the H2O2-dependent benzylamine-stimulated glucose transport. This indicates that various phenolic compounds block downstream effects of H2O2 produced by biogenic or exogenous amine oxidation without directly inhibiting AO. Phenolic compounds remain of interest regarding their capacity to limit oxidative stress rather than inhibiting AO.

Brain Formaldehyde is Related to Water Intake behavior.

A promising strategy for the prevention of Alzheimer’s disease (AD) is the identification of age-related changes that place the brain at risk for the disease. Additionally, AD is associated with chronic dehydration, and one of the significant changes that are known to result in metabolic dysfunction is an increase in the endogenous formaldehyde (FA) level. Here, we demonstrate that the levels of uric formaldehyde in AD patients were markedly increased compared with normal controls. The brain formaldehyde levels of wild-type C57 BL/6 mice increased with age, and these increases were followed by decreases in their drinking frequency and water intake. The serum arginine vasopressin (AVP) concentrations were also maintained at a high level in the 10-month-old mice. An intravenous injection of AVP into the tail induced decreases in the drinking frequency and water intake in the mice, and these decreases were associated with increases in brain formaldehyde levels. An ELISA assay revealed that the AVP injection increased both the protein level and the enzymatic activity of semicarbazide-sensitive amine oxidase (SSAO), which is an enzyme that produces formaldehyde. In contrast, the intraperitoneal injection of formaldehyde increased the serum AVP level by increasing the angiotensin II (ANG II) level, and this change was associated with a marked decrease in water intake behavior. These data suggest that the interaction between formaldehyde and AVP affects the water intake behaviors of mice. Furthermore, the highest concentration of formaldehyde in vivo was observed in the morning. Regular water intake is conducive to eliminating endogenous formaldehyde from the human body, particularly when water is consumed in the morning. Establishing good water intake habits not only effectively eliminates excess formaldehyde and other metabolic products but is also expected to yield valuable approaches to reducing the risk of AD prior to the onset of the disease.

Short-term and rapid effects of lysophosphatidic acid on human adipose cell lipolytic and glucose uptake activities

Lysophosphatidic acid (LPA) is a bioactive phospholipid that activates cell proliferation, differentiation and migration via the activation of its membrane-bound receptors (LPAR 1 to 6) expressed in various tissues and organs. Adipose tissue produces LPA, which, in turn, increases preadipocyte proliferation, mainly through the stimulation of LPA1R. However, while LPA plasma levels increase with obesity, only few studies have investigated the acute autocrine properties of LPA on mature adipocytes. We therefore assessed the lipolytic and antilipolytic effects of LPA on human adipocytes. Here, we show that, in human subcutaneous adipocytes, LPA (0.1–10 µM) did not mimic insulin effects in human adipocytes, i.e. lipolysis inhibition and glucose uptake activation. By contrast, supramicromolar doses of the phospholipid slightly activated lipolysis, and the effect of 100 µM LPA was additive to the β-adrenergic stimulation of lipolysis by isoprenaline. Moreover, LPA did not alter the activity of primary amine oxidase, an enzyme highly expressed in human adipose cells. Our observations indicate that, although rapid and direct, LPA impact on triglyceride storage in mature adipocytes is less pronounced than its ability to stimulate proliferation in preadipocytes.

Interstitial renal fibrosis due to multiple cisplatin treatments is ameliorated by semicarbazide-sensitive amine oxidase inhibition

Elucidation of acute kidney diseases and disorders (AKD), including acute kidney injury (AKI), is important to prevent their progression to chronic kidney disease. Current animal AKI models are often too severe for use in evaluating human AKI. Therefore, new animal models of mild kidney injury are needed. Here a new clinically relevant animal model using multiple low doses of cisplatin (CP) was used to evaluate AKD. When 10 mg/kg CP was administered intraperitoneally once weekly for three times to L-type fatty acid-binding protein (L-FABP) transgenic mice, moderate renal interstitial fibrosis and tubule dilatation occurred, accompanied by brush-border loss. Urinary L-FABP, a promising biomarker of AKI, changed more drastically than blood urea nitrogen or creatinine. Preventing fibrosis in organs was also studied. Oral administration of a recently reported selective semicarbazide-sensitive amine oxidase inhibitor, PXS-4728A, for 1 week attenuated kidney injury and interstitial fibrosis compared with vehicle. Inhibition of renal lipid accumulation in semicarbazide-sensitive amine oxidase inhibitor-treated mice, together with reduced oxidative stress and L-FABP suppression in proximal tubules, suggested an antifibrotic effect of semicarbazide-sensitive amine oxidase inhibition in this CP-AKD model, a representative onco-nephrology. Thus, semicarbazide-sensitive amine oxidase inhibitors may be promising candidates for the prevention of chronic kidney disease in patients using CP to treat malignancy.

Microarray studies on the effect of silencing tynA in Escherichia coli K-12.

To study the biological role of the tynA gene product of Escherichia coli, a primary amine oxidase (ECAO, E.C. 1.4.3.21), the tynA gene was genetically silenced by conjugation with a kanamycin resistance cassette. We used a microarray method to compare the mRNA expression in the modified strain (ΔtynA) to that in the wild type (wt) strain at the time of induction of ECAO expression (0 h) as well as 1 h and 4 h after the induction. These data in brief describe the different experimental conditions, sample preparation, data collection and analysis of the conducted microarray experiment. The differential expression of genes in the studied strains 1 h after the induction of ECAO expression is described. The microarray data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE65385.

Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate.

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H2O2 was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H2O2, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.

Disruption of phactr-1 pathway triggers pro-inflammatory and pro-atherogenic factors: New insights in atherosclerosis development.

Significant interest has recently emerged for phosphatase and actin regulatory protein (PHACTR1) gene in heart diseases prognosis. However, the functional role of phactr-1 protein remains elusive in heart related-diseases such as atherosclerosis, coronary artery calcification, ischaemic stroke, coronary artery stenosis and early-onset myocardial infarction. Phactr-1 is directly regulated by vascular endothelial growth factor A165 (VEGF-A165) through VEGF receptor 1 (VEGR-1) and Neuropilin-1 (NRP-1). Using an antagonist peptide approach to inhibit the interaction of VEGF-A165 to NRP-1 and VEGF-R1, we highlighted the importance of both cysteine residues located at the end of VEGF-A165 exon-7 and at the exon-8 to generate functional peptides, which decreased Phactr-1 expression. Here, we report original data showing Phactr-1 down-expression induces the expression of Matrix Metalloproteinase (MMP) regulators such as Tissue inhibitor of metalloproteinase (TIMP-1/-2) and Reversion-inducing-cysteine-rich protein with kazal motifs (RECK). Furthermore, focal adhesion kinases (FAK/PYK2/PAXILLIN) and metabolic stress (AMPK/CREB/eNOS) pathways were inhibited in endothelial cells. Moreover, the decrease of phactr-1 expression induced several factors implicated in atherosclerotic events such as oxidized low-density lipoprotein receptors (CD36, Clusterin, Cadherin-13), pro-inflammatory proteins including Thrombin, Thrombin receptor 1 (PAR-1), A Disintegrin And Metalloprotease domain-9/-17 (ADAM-9/-17), Trombospondin-2 and Galectin-3. Besides, Phactr-1 down-expression also induces emerging atherosclerosis biomarkers such as semicarbazide-sensitive amine oxidase (SSAO) and TGF-beta-inducible gene h3 (βIG-H3). In this report, we show for the first time the direct evidence of the phactr-1 biological function in the regulation of pro-atherosclerotic molecules. This intriguing result strengthened heart diseases PHACTR-1 single-nucleotide polymorphisms (SNP) correlation. Taken together, our result highlighted the pivotal role of phactr-1 protein in the pathogenesis of atherosclerosis.

Abstract P626: Amine Oxidase Activity in Mesenteric Perivascular Adipose Tissue Adipocytes is Mediated by Semicarbazide Sensitive Amine Oxidase

Perivascular adipose tissue (PVAT) is important in regulating vascular tone and reduces contraction of vessels to various agonists. PVAT is composed of adipocytes and the stromal vascular fraction (SVF), which contains immune cells, neurons, endothelial cells, fibroblasts and preadipocytes. Adipocytes, the main cell type in PVAT, contain various amine oxidases. We hypothesized that amine oxidases in mesenteric PVAT would metabolize vasoactive amines. To determine the primary enzyme(s) responsible for the amine oxidase activity in PVAT, an Amplex Red assay was performed. We added tyramine, a substrate for monoamine oxidase-A & B (MAO-A, MAO-B) and semicarbazide sensitive amine oxidase (SSAO), to cell and tissue fractions from male Sprague Dawley rats to test for H 2 O 2 production as an indicator of oxidase activity. Oxidase activity in mesenteric PVAT increased from 7.92±2.27 (vehicle) to 20.83±5.30 with the addition of tyramine (p<0.05) and from 9.30±2.55 to 24.60±6.38 in adipocytes (p<0.05; pmol/min·mg±SEM; N=5). In both the mesenteric PVAT and isolated adipocytes, the oxidase activity was reduced to baseline levels (vehicle) by pre-incubating with the SSAO inhibitor semicarbazide (1 mM; PVAT: 8.49±2.56, adipocytes: 9.43±3.21; p<0.05; N=4-5). No reduction in oxidase activity in both the mesenteric PVAT and the isolated adipocytes was observed when the MAO-A inhibitor clorgyline or the MAO-B inhibitor pargyline (1 μM; a concentration at which each drug is specific for their respective enzyme) were used. Westerns revealed that mesenteric PVAT adipocytes had the highest expression of monomeric SSAO (~80 kDa; 124.75±30.71%) compared to the whole PVAT (66.15±9.92%), blood vessels (48.2±6.89%) and the SVF (38.76±11.65%) as quantified by percent density relative to β-actin using protein isolated from male Sprague Dawley rats (p<0.05; values given as %β-actin±SEM, N=4). Oxidase activity within the SVF remains to be determined. These findings support that most of the oxidase activity in mesenteric PVAT can be attributed to SSAO and the adipocyte is an abundant source of SSAO. Due to the proximity of PVAT to the blood vessel, oxidase activity in PVAT through SSAO may alter vascular tone by metabolizing vasoactive amines and through the production of H 2 O 2 .

Hypoxia inhibits semicarbazide-sensitive amine oxidase activity in adipocytes

Semicarbazide-sensitive amine oxidase (SSAO), an enzyme highly expressed on adipocyte plasma membranes, converts primary amines into aldehydes, ammonium and hydrogen peroxide, and is likely involved in endothelial damage during the course of diabetes and obesity. We investigated whether in vitro, adipocyte SSAO was modulated under hypoxic conditions that is present in adipose tissue from obese or intensive care unit. Physical or pharmacological hypoxia decreased SSAO activity in murine adipocytes and human adipose tissue explants, while enzyme expression was preserved. This effect was time-, dose-dependent and reversible. This down-regulation was confirmed in vivo in subcutaneous adipose tissue from a rat model of hypoxia. Hypoxia-induced suppression in SSAO activity was independent of the HIF-1-α pathway or of oxidative stress, but was partially antagonized by medium acidification. Hypoxia-induced down-regulation of SSAO activity could represent an adaptive mechanism to lower toxic molecules production, and may thus protect from tissue injury during these harmful conditions.

Determination of human serum semicarbazide-sensitive amine oxidase activity via flow injection analysis with fluorescence detection after online derivatization of the enzymatically produced benzaldehyde with 1,2-diaminoanthraquinone

A fast, simple, and sensitive flow injection analysis method was developed for the measurement of semicarbazide-sensitive amine oxidase (SSAO) activity in human serum. Benzaldehyde, generated by the action of SSAO after incubation of serum with benzylamine, was derivatized with a novel aromatic aldehyde-specific reagent (1,2-diaminoanthraquinone) and the fluorescent product was measured by fluorescence detection at excitation and emission wavelengths of 390 and 570nm, respectively. Serum SSAO activity was defined as benzaldehyde (nmol) formed per milliliter serum per hour. The method was linear over SSAO activity of 0.2–150.0nmolmL−1h−1 with a detection limit of 0.06nmolmL−1h−1. The %RSD of intra-day and inter-day precision did not exceed 9.4% and the accuracy ranged from −6.5 to −0.6%. The method was applied for the determination of the serum SSAO activity in healthy controls (C, n=24) and diabetes mellitus patients (DM, n=18). It was demonstrated that the activity (mean±SE) of SSAO in diabetics sera was significantly higher than that in healthy subjects’ ones (DM; 73.3±1.8nmolmL−1h−1 vs C; 58.9±2.2nmolmL−1h−1, P<0.01).

Isolation of Lysyl Oxidase from Human Umbilical Cord

Five isoforms of human lysyl oxidases (LOX, LOXL1‐4) catalyze oxidative deamination of various primary amines and ε‐amino groups of lysine residues in proteins. LOX expression is up‐regulated by hypoxia in normal and cancer tissues. Usually, LOX is isolated from bovine aorta. However, authentic human enzyme should be preferred for pharmacological studies. We have optimized the aorta LOX protocol for human umbilical cord. The procedure includes homogenization, differential washes, urea extraction, absorption on hydroxyapatite and sulfoethylcellulose, followed by chromatography on DEAE‐cellulose and gel filtration. Simultaneously, semicarbazide sensitive amine oxidase (SSAO) can be also isolated using triton extraction followed by concanavalin A and ion exchange chromatography. Amine oxidase activities were measured as hydrogen peroxide release coupled to oxidation of 10‐acetyl‐3,7‐dihydrophenoxazine by horseradish peroxidase with methylamine as a SSAO substrate and free lysine or polyallylamine for LOX. In contrast with placenta, the cord lacks monoamine oxidase and diamine oxidase. In comparison with aorta, umbilical cord is poor in SSAO, whereas the yield of active lysyl oxidase is several folds higher. Immunoblotting using polyclonal antibodies against the five lysyl oxidase isoforms indicate that the isolated preparation contains predominantly LOX. In conclusion, the human umbilical cord is an excellent source of lysyl oxidase. Supported by RFBR grant 13‐04‐01413.

Prenatal dietary load of Maillard reaction products combined with postnatal Coca-Cola drinking affects metabolic status of female Wistar rats.

AimTo assess the impact of prenatal exposure to Maillard reaction products (MRPs) -rich diet and postnatal Coca-Cola consumption on metabolic status of female rats. Diet rich in MRPs and consumption of saccharose/fructose sweetened soft drinks is presumed to impose increased risk of development of cardiometabolic afflictions, such as obesity or insulin resistance.MethodsAt the first day of pregnancy, 9 female Wistar rats were randomized into two groups, pair-fed either with standard rat chow (MRP-) or MRPs-rich diet (MRP+). Offspring from each group of mothers was divided into two groups and given either water (Cola-) or Coca-Cola (Cola+) for drinking ad libitum for 18 days. Oral glucose tolerance test was performed, and circulating markers of inflammation, oxidative stress, glucose and lipid metabolism were assessed.ResultsMRP+ groups had higher weight gain, significantly so in the MRP+/Cola- vs MRP-/Cola-. Both prenatal and postnatal intervention increased carboxymethyllysine levels and semicarbazide-sensitive amine oxidase activity, both significantly higher in MRP+/Cola + than in MRP-/Cola-. Total antioxidant capacity was lower in MRP+ groups, with significant decrease in MRP+/Cola + vs MRP-/Cola+. Rats drinking Coca-Cola had higher insulin, homeostatic model assessment of insulin resistance, heart rate, advanced oxidation of protein products, triacylglycerols, and oxidative stress markers measured as thiobarbituric acid reactive substances compared to rats drinking water, with no visible effect of MRPs-rich diet.ConclusionMetabolic status of rats was affected both by prenatal and postnatal dietary intervention. Our results suggest that combined effect of prenatal MRPs load and postnatal Coca-Cola drinking may play a role in development of metabolic disorders in later life.