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Biomedical subjects

Karsten Sydow

Publications and source records attributed to Karsten Sydow.

At least 19 recordsLinked to original sources
PubMed2006

Clopidogrel improves systemic endothelial nitric oxide bioavailability in patients with coronary artery disease: evidence for antioxidant and antiinflammatory effects.

BACKGROUND: Platelet stimulation and activation are known not only as prerequisite of clot formation but are increasingly recognized as important contributors to inflammation and vascular injury. The present study in patients with symptomatic coronary disease investigated whether platelet adenosine diphosphate receptor blockade by clopidogrel exerts beneficial effects on endothelial nitric oxide bioavailability, oxidative stress, and/or inflammatory status. METHODS AND RESULTS: One hundred three consecutive patients with symptomatic coronary disease and long-term aspirin therapy were studied. Endothelium-dependent and -independent vasodilation was determined measuring forearm blood flow (FBF)-responses to acetylcholine with and without N(G)-monomethyl-L-arginin (L-NMMA) and sodium nitroprusside, by using venous occlusion plethysmography. Patients were randomized to receive additional treatment with clopidogrel or placebo. Vascular function tests were repeated after 5 weeks and showed significant improvement of acetylcholine-induced vasodilatation and L-NMMA responses in the clopidogrel-added group (max. FBF from 9.8+/-0.3 to 14.7+/-0.4; L-NMMA-response from 3.7+/-0.1 to 6.8+/-0.3 mL/100 mL/min). In contrast, no significant changes were observed in the placebo group. Sodium nitroprusside-induced vasodilation was not changed in either group. Urinary excretion of 8-iso-prostaglandin F2alpha and plasma levels of hsCRP, sCD40L, and RANTES were reduced in patients on additional treatment with clopidogrel, but not in patients on placebo. CONCLUSIONS: Clopidogrel improves endothelial nitric oxide bioavailability and diminishes biomarkers of oxidant stress and inflammation in patients with symptomatic coronary artery disease, suggesting that beyond inhibition of platelet aggregation, adenosine phosphate receptor blockade may also have promising vasoprotective effects.

Acetylcholine↗
PubMed2006

Heparins increase endothelial nitric oxide bioavailability by liberating vessel-immobilized myeloperoxidase.

BACKGROUND: Neutrophils and monocytes are centrally linked to vascular inflammatory disease, and leukocyte-derived myeloperoxidase (MPO) has emerged as an important mechanistic participant in impaired vasomotor function. MPO binds to and transcytoses endothelial cells in a glycosaminoglycan-dependent manner, and MPO binding to the vessel wall is a prerequisite for MPO-dependent oxidation of endothelium-derived nitric oxide (NO) and impairment of endothelial function in animal models. In the present study, we investigated whether heparin mobilizes MPO from vascular compartments in humans and defined whether this translates into increased vascular NO bioavailability and function. METHODS AND RESULTS: Plasma MPO levels before and after heparin administration were assessed by ELISA in 109 patients undergoing coronary angiography. Whereas baseline plasma MPO levels did not differ between patients with or without angiographically detectable coronary artery disease (CAD), the increase in MPO plasma content on bolus heparin administration was higher in patients with CAD (P=0.01). Heparin treatment also improved endothelial NO bioavailability, as evidenced by flow-mediated dilation (P<0.01) and by acetylcholine-induced changes in forearm blood flow (P<0.01). The extent of heparin-induced MPO release was correlated with improvement in endothelial function (r=0.69, P<0.01). Moreover, and consistent with this tenet, ex vivo heparin treatment of extracellular matrix proteins, cultured endothelial cells, and saphenous vein graft specimens from CAD patients decreased MPO burden. CONCLUSIONS: Mobilization of vessel-associated MPO may represent an important mechanism by which heparins exert antiinflammatory effects and increase vascular NO bioavailability. These data add to the growing body of evidence for a causal role of MPO in compromised vascular NO signaling in humans.

Aged↗
PubMed2005

Dimethylarginine dimethylaminohydrolase overexpression suppresses graft coronary artery disease.

BACKGROUND: Graft coronary artery disease (GCAD) is the leading cause of death after the first year of heart transplantation. The reduced bioavailability of endothelium-derived nitric oxide (NO) may play a role in endothelial vasodilator dysfunction and the structural changes that are characteristic of GCAD. A potential contributor to endothelial pathobiology is asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor. We hypothesized that lowering ADMA concentrations by dimethylarginine dimethylaminohydrolase (DDAH) overexpression in the recipient might suppress GCAD and long-term immune responses in murine cardiac allografts. METHODS AND RESULTS: In one series, donor hearts of C-H-2(bm12)KhEg (H-2(bm12)) wild-type (WT) mice were heterotopically transplanted into C57BL/6 (H-2b) transgenic mice overexpressing human DDAH-I or WT littermates and procured after 4 hours of reperfusion (WT and DDAH-I recipients, n=6 each). In a second series, donor hearts were transplanted into DDAH-I-transgenic or WT mice and procured 30 days after transplantation (n=7 each). In DDAH-I recipients, plasma ADMA concentrations were lower, in association with reduced myocardial generation of superoxide anion (WT versus DDAH-I, 465.7+/-79.8 versus 173.4+/-32.3 micromol.L(-1).mg(-1).h(-1); P=0.02), inflammatory cytokines, adhesion molecules, and chemokines. GCAD was markedly reduced in cardiac allografts of DDAH-I-transgenic recipients as assessed by luminal narrowing (WT versus DDAH, 79+/-2% versus 33+/-7%; P<0.01), intima-media ratio (WT versus DDAH, 1.1+/-0.1 versus 0.5+/-0.1; P<0.01), and the percentage of diseased vessels (WT versus DDAH, 100+/-0% versus 62+/-10%; P<0.01). CONCLUSIONS: Overexpression of DDAH-I attenuated oxidative stress, inflammatory cytokines, and GCAD in murine cardiac allografts. The effect of DDAH overexpression may be mediated by its reduction of plasma and tissue ADMA concentrations.

Amidohydrolases↗
PubMed2005

Elevation of asymmetric dimethylarginine in patients with unstable angina and recurrent cardiovascular events.

AIMS: We investigated the role of asymmetric dimethylarginine (ADMA) for clinical outcome of patients with unstable angina. METHODS AND RESULTS: Forty-five patients with stable angina, 36 patients with unstable angina, and 40 healthy controls were included in this study. Coronary artery disease (CAD) patients were prospectively followed for 1 year. ADMA levels were measured at baseline and after 6 weeks using a validated ELISA. Baseline ADMA concentration in controls was significantly lower than in patients with CAD (0.59+/-0.23 vs. 0.76+/-0.17 micromol/L; P<0.001). Patients with unstable angina had significantly higher baseline ADMA levels than patients with stable angina (0.82+/-0.18 vs. 0.73+/-0.15 micromol/L; P=0.01). There was a significant reduction of ADMA levels at 6 weeks after percutaneous coronary intervention (PCI) in patients with unstable angina who experienced no recurrent cardiovascular event (from 0.81+/-0.14 to 0.73+/-0.19 micromol/L; P<0.05). In contrast, patients with unstable angina who had an event showed no significant decrease in ADMA at 6 weeks. Actuarial survival analysis showed a significantly higher event rate in patients with persistently elevated ADMA plasma concentrations. CONCLUSION: ADMA is significantly elevated in patients with unstable angina. A reduced ADMA level at 6 weeks after PCI may indicate a decreased risk of recurrent cardiovascular events.

Adult↗
PubMed2005

Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4.

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.

Acetylcholine↗
PubMed2005

Overexpression of dimethylarginine dimethylaminohydrolase reduces tissue asymmetric dimethylarginine levels and enhances angiogenesis.

BACKGROUND: This study was designed to determine whether overexpression of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) could enhance angiogenesis by reducing levels of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). METHODS AND RESULTS: In DDAH1 transgenic (TG) and wild-type mice (each n=42), the role of DDAH overexpression on angiogenesis was studied by use of the disk angiogenesis system and a murine model of hindlimb ischemia (each n=21). After surgery, animals were treated with either PBS or the NOS inhibitors ADMA or N(omega)-nitro-L-arginine methyl ester (L-NAME; each 250 micromol x kg(-1) x d(-1)) by use of osmotic minipumps (each n=7). L-NAME was chosen to study an inhibitor that is not degraded by DDAH. Neovascularization in the disk angiogenesis system was impaired by both NOS inhibitors; however, TG animals were resistant to the effects of ADMA on neovascularization. Similarly, TG mice were more resistant to the inhibitory effect of ADMA on angioadaptation (angiogenesis and arteriogenesis) after hindlimb ischemia, as assessed by fluorescent microsphere studies and postmortem microangiograms. Enhanced neovascularization and limb perfusion in TG mice were associated with reduced plasma and tissue ADMA levels and enhanced tissue NOS enzyme activity. CONCLUSIONS: We describe a novel mechanism by which DDAH regulates postnatal neovascularization. Therapeutic manipulation of DDAH expression or activity may represent a novel approach to restore tissue perfusion.

Amidohydrolases↗
PubMed2005

Insulin resistance: potential role of the endogenous nitric oxide synthase inhibitor ADMA.

The insulin resistance syndrome (IRS) is considered to be a new target of risk-reduction therapy. The IRS is a cluster of closely associated and interdependent abnormalities and clinical outcomes that occur more commonly in insulin-resistant/hyperinsulinemic individuals. This syndrome predisposes individuals to type 2 diabetes, cardiovascular diseases, essential hypertension, certain forms of cancer, polycystic ovary syndrome, nonalcoholic fatty liver disease, and sleep apnea. In patients at high risk for cardiovascular diseases, endothelial dysfunction is observed in morphologically intact vessels even before the onset of clinically manifest vascular disease. Indeed, there are several lines of evidence that indicate that endothelial function is compromised in situations where there is reduced sensitivity to endogenous insulin. It is well established that a decreased bioavailability of nitric oxide (NO) contributes to endothelial dysfunction. Furthermore, NO may modulate insulin sensitivity. Activation of NO synthase (NOS) augments blood flow to insulin-sensitive tissues (i.e. skeletal muscle, liver, adipose tissue), and its activity is impaired in insulin resistance. Inhibition of NOS reduces the microvascular delivery of nutrients and blunts insulin-stimulated glucose uptake in skeletal muscle. Furthermore, induction of hypertension by administration of the NOS inhibitor NG-monomethyl-L-arginine is also associated with insulin resistance in rats. Increased levels of asymmetric dimethylarginine (ADMA) are associated with endothelial vasodilator dysfunction and increased risk of cardiovascular diseases. An intriguing relationship exists between insulin resistance and ADMA. Plasma levels of ADMA are positively correlated with insulin resistance in nondiabetic, normotensive people. New basic research insights that provide possible mechanisms underlying the development of insulin resistance in the setting of impaired NO bioavailability will be discussed.

Animals↗
PubMed2004

Oxidative stress and mitochondrial aldehyde dehydrogenase activity: a comparison of pentaerythritol tetranitrate with other organic nitrates.

Mitochondrial aldehyde dehydrogenase (ALDH-2) was recently identified to be essential for the bioactivation of glyceryl trinitrate (GTN). Here we assessed whether other organic nitrates are bioactivated by a similar mechanism. The ALDH-2 inhibitor benomyl reduced the vasodilator potency, but not the efficacy, of GTN, pentaerythritol tetranitrate (PETN), and pentaerythritol trinitrate in phenylephrine-constricted rat aorta, whereas vasodilator responses to isosorbide dinitrate, isosorbide-5-mononitrate, pentaerythritol dinitrate, pentaerythritol mononitrate, and the endothelium-dependent vasodilator acetylcholine were not affected. Likewise, benomyl decreased GTN- and PETN-elicited phosphorylation of the cGMP-activated protein kinase substrate vasodilator-stimulated phosphoprotein (VASP) but not that elicited by other nitrates. The vasodilator potency of organic nitrates correlated with their potency to inhibit ALDH-2 dehydrogenase activity in mitochondria from rat heart and increase mitochondrial superoxide formation, as detected by chemiluminescence. In contrast, mitochondrial ALDH-2 esterase activity was not affected by PETN and its metabolites, whereas it was inhibited by benomyl, GTN applied in vitro and in vivo, and some sulfhydryl oxidants. The bioactivation-related metabolism of GTN to glyceryl-1,2-dinitrate by isolated RAW macrophages was reduced by the ALDH-2 inhibitors benomyl and daidzin, as well as by GTN at concentrations >1 microM. We conclude that mitochondrial ALDH-2, specifically its esterase activity, is required for the bioactivation of the organic nitrates with high vasodilator potency, such as GTN and PETN, but not for the less potent nitrates. It is interesting that ALDH-2 esterase activity was inhibited by GTN only, not by the other nitrates tested. This difference might explain why GTN elicits mitochondrial superoxide formation and nitrate tolerance with the highest potency.

Aldehyde Dehydrogenase↗
PubMed2004

Endothelium- and nitric oxide-dependent vasorelaxing activities of gamma-butyrobetaine esters: possible link to the antiischemic activities of mildronate.

Mildronate [3-(2,2,2-trimethylhydrazine) propionate (THP)] is an antiischemic drug acting mainly via inhibition of fatty acid beta-oxidation. Some effects of the drug cannot be explained by the latter mechanism. We tested the eventual nitric oxide (NO) dependence of the mildronate action. Mildronate, gamma-butyrobetaine (GBB) and GBB methyl ester induced transient increases in nitric oxide (NO) concentrations in rat blood and myocardium. In vitro, these compounds neither modified the activities of purified neuronal and endothelial recombinant nitric oxide synthases (NOSs) nor were able to interact with their active site. GBB induced vasodilatation at high concentrations only (EC50 = 5 x 10(-5) M) while mildronate alone displayed no vasodilating effect although it enhanced the GBB vasodilating activity. GBB methyl and ethyl esters were found more potent vasodilators (EC50 = 2.5 x 10(-6) M). Pretreatment of aortic rings with NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) abolished vasodilating effects of the compounds. A hypothesis explaining NO and endothelium-dependent effects of mildronate and its analogues is proposed.

Animals↗
PubMed2004

Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012.

In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.

Animals↗
PubMed2004

Capillary electrophoretic and micellar electrokinetic separations of asymmetric dimethyl-L-arginine and structurally related amino acids: quantitation in human plasma.

We report the development of efficient electrophoretic methods for the separation and quantification of L-arginine and six naturally occurring derivatives that are structurally and functionally related. Capillary electrophoresis (CE) employing a concentrated borate buffer at pH 9.4 achieves the separation of mixtures containing dimethyl-L-arginine, NG-monomethyl-L-arginine, L-arginine, L-homoarginine, L-ornithine, and L-citrulline as 4-fluoro-7-nitrobenzofurazan derivatives. In addition, the separation of the isomeric dimethyl-L-arginine derivatives (symmetric and asymmetric) is attained with baseline resolution by micellar electrokinetic chromatography (MEKC) when a high concentration of deoxycholic acid is added as a surfactant to the same running buffer. The influence of buffer type, concentration, and pH on the separation was studied to optimize separation conditions. The limit of quantitation (LOQ) for asymmetric dimethyl-L-arginine in aqueous solution was determined to be 20 microM using UV absorption in a CE separation and 0.1 microM using laser induced fluorescence (LIF) detection in an MEKC separation. This newly developed method was successfully applied for the quantitation of asymmetric dimethyl-L-arginine and L-arginine in human plasma samples at levels that might be used as a clinical diagnostic for cardiovascular disease (0.125 microM LOQ).

4-Chloro-7-nitrobenzofurazan↗
PubMed2004

Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012.

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10-100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.

Animals↗
PubMed2004

Central role of mitochondrial aldehyde dehydrogenase and reactive oxygen species in nitroglycerin tolerance and cross-tolerance.

Recent studies suggest that mitochondrial aldehyde dehydrogenase (ALDH-2) plays a central role in the process of nitroglycerin (glyceryl trinitrate, GTN) biotransformation in vivo and that its inhibition accounts for mechanism-based tolerance in vitro. The extent to which ALDH-2 contributes to GTN tolerance (impaired relaxation to GTN) and cross-tolerance (impaired endothelium-dependent relaxation) in vivo remain to be elucidated. Rats were treated for three days with GTN. Infusions were accompanied by decreases in vascular ALDH-2 activity, GTN biotransformation, and cGMP-dependent kinase (cGK-I) activity. Further, whereas in control vessels, multiple inhibitors and substrates of ALDH-2 reduced both GTN-stimulation of cGKI and GTN-induced vasodilation, these agents had little effect on tolerant vessels. A state of functional tolerance (in the GTN/cGMP pathway) was recapitulated in cultured endothelial cells by knocking down mitochondrial DNA (rho(0) cells). In addition, GTN increased the production of reactive oxygen species (ROS) by mitochondria, and these increases were associated with impaired relaxation to acetylcholine. Finally, antioxidants/reductants decreased mitochondrial ROS production and restored ALDH-2 activity. These observations suggest that nitrate tolerance is mediated, at least in significant part, by inhibition of vascular ALDH-2 and that mitochondrial ROS contribute to this inhibition. Thus, GTN tolerance may be viewed as a metabolic syndrome characterized by mitochondrial dysfunction.

Aldehyde Dehydrogenase↗
PubMed2004

Endothelial dysfunction in patients with peripheral arterial disease and chronic hyperhomocysteinemia: potential role of ADMA.

Hyperhomocysteinemia is associated with an enhanced risk for cardiovascular disease. Patients with peripheral arterial disease (PAD) show an increased prevalence of hyperhomocysteinemia. A decreased biological activity of nitric oxide (NO) may contribute to homocysteine-associated endothelial dysfunction. This study was designed to investigate whether elevated levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) are involved in endothelial dysfunction in patients with chronic hyperhomocysteinemia and PAD. A total of 76 patients (58 males and 18 females; mean age 65.2 +/- 2.0 years) with PAD were included in the analysis and characterized according to demographic variables and cardiovascular risk factors. Flow-dependent vasodilation (FDD) was determined by high-resolution ultrasound in the radial artery. Total plasma homocysteine (plasma tHcy) and ADMA levels were measured by HPLC. Urinary nitrate was quantified using gas chromatography-mass spectrometry. Patients with plasma tHcy in the highest tertile (n = 27; i.e. > 10.6 micromol/l) had a mean plasma level of 14.4 +/- 1.21 mol/l compared with 9.9 +/- 0.1 micromol/l in those patients in the middle tertile (n = 22; p < 0.05) and 9.4 +/- 0.1 micromol/l in those in the lowest tertile (n = 27; i.e. <9.6 micromol/l; p < 0.05). The hyperhomocysteinemic individuals (highest tertile) had a significantly decreased FDD compared with healthy age-matched controls (n = 15) (7.6 +/- 1.0 vs 13.0 +/- 0.4%; p < 0.05), higher plasma ADMA concentrations (4.0 +/- 0.3 vs 2.6 +/- 0.3 micromol/l; p < 0.05), and a lower urinary nitrate excretion rate (89.5 +/- 13.4 vs 131.3 +/- 17.9 micromol/mmol creatinine; p < 0.05) compared with patients with plasma tHcy in the lowest tertile. Multivariate regression analysis including plasma tHcy, ADMA, total cholesterol, diabetes mellitus, smoking, and systolic blood pressure revealed ADMA as the only significant factor determining FDD (p < 0.05). In conclusion, we demonstrated a stronger relationship between impaired endothelial function and elevated ADMA levels in comparison with plasma tHcy concentrations in patients with PAD and chronic hyperhomocysteinemia. This may raise the question of whether different therapeutical options that interact indirectly with plasma tHcy, i.e. treatment with ACE inhibitors and AT1-receptor blockers to reduce ADMA plasma concentrations or L-arginine, could be a beneficial tool for treating patients with hyperhomocysteinemia.

Adult↗
PubMed2004

Determination of asymmetric dimethylarginine (ADMA) using a novel ELISA assay.

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Elevated ADMA plasma levels have been reported in connection with diseases associated with an impaired endothelial L-arginine-NO pathway and endothelial dysfunction, such as atherosclerosis, hypercholesterolemia, chronic heart failure, diabetes mellitus, and hypertension. NO production by NOS is decreased due to elevated ADMA levels. In fact, there is increasing interest in determination of ADMA levels in samples of various origins. The aim of this work was to develop a precise and easy immunoassay in contrast to the existing methods, such as HPLC, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC)-MS. We determined cross-reactivity in our immunoassay of 1.2% for symmetric dimethylarginine and <0.02% for L-arginine. The limit of quantitation was 0.05 micromol/l. We found good correlation of the values measured when we compared our assay with LC-tandem MS (n = 29; r = 0.984; p < 0.0001). We determined ADMA levels in human serum and plasma, mouse and rat plasma, and cell culture supernatant. For human plasma we found a mean of 0.65 micromol/l in healthy subjects. In the plasma of mice and rats we found mean concentrations of 1.05 and 1.09 micromol/l, respectively.

Animals↗
PubMed2003

Does nitric oxide mediate the vasodilator activity of nitroglycerin?

Nitroglycerin (glyceryl trinitrate, GTN) relaxes blood vessels primarily via activation of the soluble guanylyl cyclase (sGC)/cGMP/cGMP-dependent protein kinase (cGK-I) pathway. Although the precise mechanism of sGC activation by GTN in the vascular wall is unknown, the mediatory role of nitric oxide (NO) has been postulated. We tested the GTN/NO hypothesis in different types of isolated rat and rabbit blood vessels using two novel approaches: (1) EPR spin trapping using colloid Fe(DETC)2 and (2) analysis of cGK-I-dependent phosphorylation of the vasodilator-stimulated phosphoprotein at Ser239 (P-VASP). For comparison, another organic nitrate, isosorbide dinitrate (ISDN), and endothelium-dependent vasodilator, calcium ionophore A23187, were tested. We found a marked discrepancy between GTN's strong vasoactivity (vasodilation and augmentation of P-VASP) and its poor NO donor properties. In aortas precontracted with phenylephrine, GTN, ISDN, and A23187 induced nearly full relaxations (>80%) and doubling of vascular P-VASP content at concentrations of 100 nmol/L, 100 micromol/L, and 1 micromol/L, respectively. GTN applied in vasorelaxant concentrations (10 to 1000 nmol/L) did not significantly increase the basal vascular NO production, in contrast to ISDN and A23187. The absence of GTN-derived NO was confirmed in rabbit vena cava and renal artery. A significant increase in vascular NO formation was observed only at suprapharmacological GTN concentrations (>10 micromol/L). The concentration dependency of NO formation from GTN was comparable to that of ISDN, although the latter exhibits 100-folds lower vasorelaxant potency. We conclude that GTN activates the sGC/cGMP/cGK-I pathway and induces vasorelaxation without intermediacy of the free radical NO. The full text of this article is available online at http://www.circresaha.org.

Animals↗
PubMed2003

ADMA and oxidative stress are responsible for endothelial dysfunction in hyperhomocyst(e)inemia: effects of L-arginine and B vitamins.

OBJECTIVES: Hyperhomocyst(e)inemia is a risk factor for atherosclerotic vascular disease, and it is associated with endothelial dysfunction. Mechanisms responsible for endothelial dysfunction in hyperhomocyst(e)inemia may involve impaired bioavailability of NO, possibly secondary to accumulation of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) and increased oxidative stress. We investigated whether oral treatment with B vitamins or L-arginine normalizes endothelium-dependent, flow-dependent vasodilation (FDD) in patients with peripheral arterial occlusive disease (PAOD) and hyperhomocyst(e)inemia. METHODS: 27 patients with PAOD and hyperhomocyst(e)inemia were assigned to oral treatment with combined B vitamins (folate, 10 mg; vitamin B-12, 200 microg; vitamin B-6, 20 mg/day), L-arginine (24 g/day) or placebo, for 8 weeks in a double-blind fashion. FDD was determined by high-resolution ultrasound in the radial artery. RESULTS: Vitamin B supplementation significantly lowered plasma homocyst(e)ine concentration from 15.8+/-1.8 to 8.7+/-1.1 micromol/l (P<0.01). However, B vitamins had no significant effect on FDD (baseline, 7.8+/-0.7%, B vitamins, 8.3+/-0.9%, placebo 8.9+/-0.7%; P=n.s.). In contrast, L-arginine treatment did not affect homocyst(e)ine levels, but significantly improved FDD (10.2+/-0.2%), probably by antagonizing the impact of elevated ADMA concentration (3.8+/-0.3 micromol/l) and reducing the oxidative stress by lowering urinary 8-iso-prostaglandin F(2alpha) (baseline, 76.3+/-7.1 vs. 62.7+/-8.3 pmol/mmol creatinine after 8 weeks). CONCLUSIONS: Oral supplementation with combined B vitamins during 8 weeks does not improve endothelium-dependent vasodilation in PAOD patients with hyperhomocyst(e)inemia, whereas L-arginine significantly improved endothelial function in these patients. Thus, accumulation of ADMA and increased oxidative stress may underlie endothelial dysfunction under hyperhomocyst(e)inemic conditions. These findings may have importance for evaluation of homocyst(e)ine-lowering therapy.

Aged↗