Marys Medicine


OS, and plasma lipids Role of raloxifene on platelet metabolism and
plasma lipids
L. Nanetti, A. Camilletti, C. M. Francucci, A. Vignini, F. Raffaelli, L. Mazzanti and M. Boscaro
Università Politecnica delle Marche, Ancona, Italy Background This study was performed to understand the metabolic effects of raloxifene, a selective oestrogen
receptor modulator, on platelets in healthy non-obese postmenopausal women. The data were compared to
untreated subjects.
Materials and methods Platelet nitric oxide activity (NO) and peroxynitrite level, platelet inducible and
endothelial nitric oxide synthase expression and plasma lipids were evaluated at baseline and after 12 months
of raloxifene or placebo treatment.
Results A significant increase of platelet NO and reduction of platelet peroxynitrite levels, as well as a decrease
of inducible nitric oxide synthase expression, was observed 12 months after raloxifene therapy as compared to
baseline or placebo treatment. Moreover, raloxifene treatment caused a significant increase in high-density
lipoprotein cholesterol and a decrease of total cholesterol and low-density lipoprotein cholesterol were observed
versus baseline values (P < 0·05). A significant positive correlation was observed between high-density lipoprotein
cholesterol and platelet NO (r = 0·76, P < 0·005) in the raloxifene group.
Conclusion Our results showed that raloxifene improves platelet metabolism in healthy postmenopausal women
through an increase of the bioavailability of platelet NO by a reduction of iNOS and the beneficial effects on lipid
metabolism. This mechanism of action of raloxifene on platelet activity may explain some cardiovascular protective
effects of this selective oestrogen receptor modulator.
Keywords Nitric oxide, NOS, peroxynitrite, platelets, postmenopausal women, raloxifene.
Eur J Clin Invest 2008; 38 (2): 117–125 Observational studies, as well as prospective randomized trials, cyclic guanosine monophosphate (cGMP) production in human reveal that the incidence of coronary artery disease is lower in platelets leading to the activation of protein kinase C and the premenopausal women than in age-matched men and inhibition of platelet aggregation [3]. NO-induced inhibition of postmenopausal women. These differences could be attributed platelet aggregation involves a decrease of the intraplatelet Ca2+ to the oestrogens that seem to exert important physiological concentration [4].
functions on endothelium-derived relaxing factor, smooth muscle The most important NOS isoform within the cardiovascular proliferation, neurons and blood elements such as platelets and system is the endothelial nitric oxide synthase (eNOS), that is leukocytes [1].
regulated by Ca2+/calmodulin [5]. On the contrary, the inducible Platelets play a vital role in vascular haemostasis. Their ability nitric oxide synthase (iNOS) is produced in response to cytokines. to aggregate and form a haemostatic plug must be carefully Under conditions of limiting l-arginine or favouring superoxide balanced with the necessity to maintain the fluid state of the anion ‘bursts', such as ischaemia-reperfusion, inflammation, blood and to avoid thrombosis. Platelet activation and recruitment hypercholesterolaemia, and angiotensin-induced hypertension, are tightly regulated by products of the endothelium, including iNOS is expressed more than eNOS and it seems to produce prostacyclin and nitric oxide (NO) [2].
superoxide ( O⋅−) and NO at comparable rates [6]. This may have NO is the only known endogenous formed radical acting as a potentially deleterious consequences as O⋅− and NO react to form, signalling messenger. It is formed by nitric oxide synthases (NOS), quenched by intraplatelet glutathione, peroxynitrite (ONOO–), which convert l-arginine to citrulline and NO. NO stimulates a very potent oxidant. Both NOS have been identified in human European Journal of Clinical Investigation Vol 38 117 L. NANETTI ET AL.
platelets and megakaryocytoid cells [7]. In normal conditions, the beginning of the study and who had a T score bone mineral platelets do not express iNOS isoform, while they do express density < –2 DS at spine (L2–L4), without a history of fractures, eNOS isoform that produces more NO than O⋅− . were eligible to participate in the trial. Informed consent was iNOS-dependent peroxynitrite production has been recently obtained from all participants and the study was approved implicated in the pathophysiology of atherosclerosis, by the Local Ethics Committee. Patients were recruited from hypercholesterolaemia, hypertension and diabetes mellitus.
those reporting to the Centre for Osteoporosis and Menopause Several studies have shown that high plasma peroxynitrite of the Unit of Endocrinology of the Department of Internal concentration promotes platelet aggregation, while low Medicine of Marche Politecnical University.
peroxynitrite plasma concentration inhibits collagen-induced Exclusion criteria were: history of cardiovascular or aggregation [8].
cerebrovascular disease, deep-vein thrombosis or pulmonary Hypercholesterolaemia induces an inhibition of embolism, bilateral oophorectomy, neoplasia, renal or hepatic endothelium-dependent vasorelaxation often referred to as dysfunction, malabsorption syndrome, smoking habits, endothelial dysfunction, which is one of the earliest abuse of alcohol or drugs, current use of cholesterol-lowering manifestations of cardiovascular disease [9]. The pathogenesis medication and HRT and cardiovascular medication. Moreover, of endothelial dysfunction involves a variety of oxygen-containing patients were required not to have used any non-steroidal radicals and of non-radical small molecules with a more or less anti-inflammatory drugs or any other drugs for at least strong oxidative capacity [10] such as superoxide, hydrogen 6 months prior to, and during, study enrolment. This peroxide, hypochloride, NO and peroxynitrite. In platelets, prospective, randomised, double-blind and placebo-controlled hypercholesterolaemia induces a hyper-reactive state which (CG) trial was performed on 80 healthy postmenopausal significantly contributes to enhanced arterial thrombus women. Postmenopausal status was defined by a follicle- formation through a reduced anti-aggregatory activity of stimulating hormone (FSH) level of more than 40 IU L–1 prostacyclin, an increased serotonin release, and an enhanced and a serum oestradiol level of less than 91·8 pmol L–1.
response to platelet aggregators such as collagen and Forty postmenopausal women were treated with RLX thromboxane [11].
60 mg day–1 for 1 year and 40 postmenopausal women Controversial results of hormone replacement therapy (HRT) were recruited as a control group (CG). Patients and controls have radically altered the contemporary understanding of its were matched by age, body mass index (BMI) and role in cardiovascular disease prevention [12]. Thus, raloxifene postmenopausal duration (years). All subjects consumed hydrochloride (RLX), a selective oestrogen receptor modulator an adequate daily calcium intake (above 1200 mg) and (SERM), should be highly desirable for postmenopausal women biochemical evaluation did not highlight hypovitaminosis [13] because of its oestrogen-agonistic effect on bone and lipid D. Moreover, participants agreed not to alter their diet and metabolism and of its oestrogen-antagonistic activity in the exercise regimes during the study protocol. All patients uterus and breasts. Moreover, SERM possesses vascular relaxing completed the study.
properties by an oestrogen receptor-dependent and Patients were asked to take one tablet or one capsule daily, NO-dependent mechanism [14].
at approximately the same time. To ensure compliance, patients The aim of the present study was to evaluate, for the first time, were asked to return the packets containing placebos or in non-obese postmenopausal women in respect to untreated medication at the end of every month.
subjects, the metabolic effects of raloxifene on platelets as they could be considered an ideal system to investigate the effects of drugs on platelet-derived NO. The following parameters were After overnight fasting, before and after 12 months of treatment, total cholesterol (TC), high-density lipoprotein cholesterol • The effects of RLX on platelet NO and peroxynitrite production; (HDL-C) and triglycerides (TG) were measured, while low- • The changes in platelet NO and peroxynitrite synthesis with the density lipoprotein cholesterol (LDL-C) was calculated by the activation of iNOS and eNOS; Friedewald formula [LDL-C = TC – HDL-C – (TG × 0·20)]. • The correlation between platelet metabolism and plasma HDL-C was measured by photometric determination by a lipoproteins in healthy postmenopausal women.
Trinder endpoint without prior separation (Direct HDL-Cholesterol, Bayer, Tarrytown, NY, USA).
Subjects and methods
Oestradiol and FSH (Automated Chemiluminiscence Systems ACS Centaur; Bayer) were measured in all patients by chemiluminescent immunometric assays. The intra- and Naturally healthy menopausal women between 56 and 64 years interassay coefficients of variation were below 5·2 and 7·3, whose last menstrual period occurred at least 5 years before 2008 The Authors. Journal Compilation 2008 Blackwell Publishing Ltd RALOXIFENE, ENOS, INOS, AND PLASMA LIPIDS the samples were sonicated. The mixture was then centrifuged Peripheral venous blood was immediately mixed with at 200 × g 5 min and the fluorescence was measured in the anticoagulant citrate dextrose pH 6·5 (ACD). Platelets were supernatant in a Perkin-Elmer LS-50B spectrofluorometer, isolated by differential centrifugation in anti-aggregation buffer at an excitation wavelength of 475 nm and emission wavelength (Tris-HCl 10 mm; NaCl 150 mm; EDTA 1 mm; glucose 5 mm; pH 7·4) according to Rao [15]. The anti-aggregation method involved a preliminary centrifugation step (200 × g for 10 min) to obtain platelet rich plasma (PRP). The platelets were then Platelet rich plasma samples were lysed in washed three times in anti-aggregation buffer and centrifuged in RadioImmunoPrecipitation Assay buffer (RIPA) lyses buffer order to remove any residual erythrocytes. A final centrifugation containing 1 × PBS, 1/100 Igepal CA-630, 0·5/100 sodium at 2000 × g for 20 min was performed to isolate the platelets, deoxycholate, 0·1/100 sodium dodecyl sulphate (SDS), which were immediately used for the experiments or stored 10 mg mL–1 Phenylmethylsulfonylfluorid (PMSF), aprotinin, at –80 °C for no longer than 15 days.
100 mmol L–1 sodium orthovanadate and 4/100 protease inhibitor cocktails by microcentrifugation at 10 000 × g for Determination of NOS activity
10 min at 4 °C. The supernatants were collected and treated NO levels were measured in the supernates of lysed with an equal volume of sample application buffer platelets, as previously described [16], using the Griess (125 mmol L–1 Tris-HCl, pH 6·8, 2/100 SDS, 5/100 glycerol, reaction [17]. Briefly, platelets were suspended in NO buffer 0·003/100 bromophenol blue, 1/100 β-mercaptoethanol). (Hepes 25 mm, NaCl 140 mm, KCl 5·4 mm, CaCl The mixture was boiled for 5 min; 15 μL of each sample was applied to each well of an 8/100 SDS polyacrylamide gel and 2 1 mm, pH 7·4) containing 1·44 mm NADPH and incubated for 1 h at 37 °C after the addition of l-arginine electrophoresed for 1 h at 130 V along with a set of molecular 100 μm. The reaction was then stopped by freeze-thawing weight markers (Broad Range, Sigma, St. Louis, MO, USA). the sample which was then sonicated. Each sample was The resolved protein bands were then transferred onto incubated for 1 h at 37 °C after the addition of nitrate polyvinylidene fluoride (PVDF) membranes at 100 V for reductase (20 mU), which reduces nitrate to nitrite. After 60 min using a transfer buffer of 25 mmol L–1 Tris base, centrifugation at 1100 × g for 15 min, the supernatant was 192 mmol L–1 glycine, and 20/100 methanol. The blots allowed to react with the Griess reagent (1% sulphanylamide/0·1% were blocked overnight at 4 °C with blocking buffer naphthylenediamine dihydrochloride/2·5% H (5/100 non-fat milk in 10 mmol L–1 Tris pH 7·5, 100 mmol L–1 chromophore absorption was read at 543 nm. Nitrite NaCl, 0·1/100 Tween 20). The blocking buffer was decanted concentration was determined by sodium nitrite in water and blots were incubated for 1 h at room temperature with as standard. NOS activity was expressed in nmol NO primary antibody rabbit anti-endothelial nitric oxide synthase produced · min–1 · mg protein–1. Protein concentration (eNOS, 1 : 1000, Chemicon, Temecula, CA, USA), rabbit was determined as described by Bradford [18] using anti-inducible NOS (iNOS, 1 : 1000, Chemicon) and rabbit albumin as standard.
antinitrotyrosine (N-Tyr 1 : 1000 Chemicon) diluted in blocking buffer. Positive controls were included Preparation of 2,7-dichlorofluorescein-free base
in all experiments as provided by the manufacturer to 2,7-dichlorofluorescein diacetate (DCFDA)-free base was confirm antibody specificity. As an internal control, blots prepared daily, by mixing 0·05 mL of 10 mmol L–1 DCFDA were re-probed with an antiβ-actin antibody (Sigma). with 2 mL of 0·01 N NaOH, at room temperature for 30 min. Blots were then washed using Tris Tween Buffer Saline The mixture was neutralized with 18·0 mL of 25 mmol L–1 (TTBS) washing buffer (10 mmol L–1 Tris pH 7·5, phosphate-buffered saline (PBS) pH 7·4. This solution was 100 mmol L–1 NaCl, 0·1/100 Tween 20) for 30 min maintained on ice in the dark until use [19].
with agitation and incubated with goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Sigma) diluted 1 : 5000 in blocking buffer Peroxynitrite production was evaluated by the use of for 1 h at room temperature followed by washes in TTBS. a fluorescence probe DCFDA as previously described. Peroxidase activity was revealed using 3,3′-diaminobenzidine Briefly, samples were incubated for 15 min with 5 μm DCFDA-free (Sigma) as a substrate.
base at 37 °C. Then the DCFDA treated samples were incubated Densitometry was performed using software AMERSHAM with or without the addition of l-arginine 100 mmol L–1 Image Master 1D. All densitometric data are expressed as mean and NG-monomethyl-l-arginine (L-NMMA) 100 mmol L–1 densities, defined as the sum of the gray values of all pixels in a for 15 min at 37 °C in the dark. After washing in PBS pH 7·4, selection divided by the number of pixels.
European Journal of Clinical Investigation Vol 38 119 L. NANETTI ET AL.
Statistical analysis was performed using the SAS statistical package (Statistical Analysis System Institute, Cary, NC, USA). Clinical characteristics of the women of both groups were compared by means of the unpaired Student's t-test. Data were compared using paired and unpaired Student's t-test. Correlations were performed by using Pearson's coefficient. All values were reported as mean ± S.D. Significance was established at the level of P < 0·05.
Clinical and biochemical characteristics
Tables 1 and 2 show clinical and biochemical data in the two randomized groups. At baseline there were no significant Figure 1 Platelet nitric oxide activity in raloxifene (RLX; n = 40)
differences between the two randomized groups in relation and placebo (CG; n = 40) treated subjects during the study. Data to age, years from menopause, blood pressure, BMI, TC, are expressed as mean ± SD, *P < 0·05 RLX vs. CG.
HDL-C, LDL-C and TG. The level of FSH and oestradiol were also not significantly different between the two groups. BMI was the same before and after RLX therapy.
As described in the literature, platelet counts (279·3 ± 74·6 vs. 265·5 ± 57·2 109 L–1, P > 0·05) were also not significantly different before and after RLX therapy [20].
NO activity and peroxynitrite level
At baseline the two groups had a similar platelet NO activity and peroxynitrite platelet level. After 12 months RLX-users showed a significant increase of platelet NOS activity compared baseline values (0·783 ± 0·040 vs. 0·348 ± 0·020 nmol · min–1· mg protein.–1; P < 0·05) and a significant decrease of peroxynitrite platelet level (102·1 ± 34 vs. 159·6 ± 14 fluorescence arbitrary Table 1 Baseline clinical characteristics on non-obese
Figure 2 Platelet peroxynitrite (Fluorescence arbitrary numbers)
postmenopausal women treated either with raloxifene (RLX) concentration in raloxifene (RLX; n = 40) and placebo (CG; or placebo (CG). Data are expressed as mean ± SD n = 40) treated subjects during the study. Data are expressed as mean ± SD, *P < 0·05 RLX vs. CG.
numbers; P < 0·05) compared with baseline values. Besides, Postmenopausal duration (years) at the end of the study, platelet NO level increased significantly Blood pressure (mmHg) in the RLX compared to controls (0·783 ± 0·040 vs. 0·380 ± 0·019 nmol nitrite · min–1·mg protein.–1; P < 0·05), while platelet peroxynitrite levels decreased significantly in the RLX group as compared to the CG (102·1 ± 34 vs. 152·6 ± 17 fluorescence arbitrary numbers; P < 0·05) (Figs 1, 2).
Oestradiol (pmol L–1) Plasma lipids
After 12 months we observed a significant increase of HDL-C
BMI, Body Mass Index (kg m–2);FSH, follicle-stimulating hormone.
and a decrease of TC and LDL-C (P < 0·05) in the RLX group 2008 The Authors. Journal Compilation 2008 Blackwell Publishing Ltd RALOXIFENE, ENOS, INOS, AND PLASMA LIPIDS Table 2 Changes in the lipoprotein profile of the enrolled healthy postmenopausal women. Values are expressed as mean ± SD
TC (mmol L–1)
HDL-C (mmol L–1)
LDL-C (mmol L–1)
TG (mmol L–1)
12 months
12 months
12 months
12 months
*P < 0·05 vs. baseline; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglycerides.
Figure 4 Correlation between platelet peroxynitrite
Figure 3 Correlation between plasma concentrations of
concentrations and platelet nitric oxide activity in 40 raloxifene high-density lipoprotein cholesterol (HDL-C) and platelet nitric treated subjects.
oxide activity in 40 raloxifene-treated subjects.
as compared to baseline values (Table 2), while TG remained societies. In myocardial infarction and unstable angina, unchanged; a significant positive correlation between HDL-C as documented by angiographical and pathological studies, and NO activity (r = 0·76; P < 0·005; Fig. 3); a significant negative platelet aggregation and then the thrombus formation within correlation between NO activity and peroxynitrite (r = –0·89; a coronary vessel are the precipitating events [21].
P < 0·0001 Fig. 4) in RLX group.
Adhesion of platelets to the endothelium is prevented by endothelial cell production of prostacyclin and NO [2]. NO eNOS and iNOS expression
is an endogenous vasodilator and anti-aggregating substance Western blot analysis using anti-iNOS and eNOS monoclonal produced in almost all human tissues.
antibodies demonstrated that both isoforms were detectable in Platelets are one potential target for oestrogen modulation platelet lysates. In fact, in RLX-users the blot density of the bands
within the cascade of factors involved in ischaemic atherosclerotic corresponding to eNOS was unchanged after 12 months, while the events. Oestrogens can modulate changes non-genomically blot density of the bands corresponding to iNOS was decreased (by affecting Ca2+ flux, lipid metabolism, etc.) [22] and after 12 months. No significant variation was highlighted in the genomically seeing that oestrogen receptors have been CG (Fig. 5a,b).
cloned from megakaryocytes and platelets [23].
Nowadays, the evidence of the efficacy of oestrogens in the prevention of coronary artery disease has been called Cardiovascular diseases remain the largest cause of morbidity into question and, consequently, great efforts have been made and mortality among postmenopausal women in Westernized to identify therapeutic alternatives such as several SERMs European Journal of Clinical Investigation Vol 38 121 L. NANETTI ET AL.
Figure 5 (a) Western immunoblots of
inducible nitric oxide synthase (iNOS);
protein expression in plasma samples
obtained from raloxifene (RLX, n = 40);
or placebo (CG, n = 40) treated subjects at
baseline and after 12 months. β-actin was
used as an internal control. (b) Western
immunoblots of endothelial nitric oxide
synthase (eNOS) protein expression in
plasma samples obtained from raloxifene
(RLX, n = 40) or placebo (CG, n = 40)
treated subjects at baseline and after
12 months. β-actin was used as an internal
control. (a,b) represent the densitometric
analysis of iNOS and eNOS bands
(P < 0·05)
2008 The Authors. Journal Compilation 2008 Blackwell Publishing Ltd RALOXIFENE, ENOS, INOS, AND PLASMA LIPIDS that would have the benefit of oestrogens without their increases the bioavailability of platelet NO. In evidence, adverse effects [24].
we have observed an inverse correlation between RLX, a benzothiophene derivative that binds with high peroxynitrite and NO that suggests a greater affinity to the oestrogen receptor [25], is one of these SERMs NOS-dependent NO bioavailability that is not used to that has been approved for the treatment of postmenopausal neutralize oxygen radicals.
osteoporosis. In postmenopausal women this drug induces Moreover, we have highlighted, in RLX-users, in accordance positive changes in the lipid profile and serum levels of with several other authors, a decrease of TC and LDL-C, an fibrinogen and homocysteine without modifying the glucose increase of HDL-C levels and unaltered TG levels [28].
metabolism and markers of inflammation [26–29]. Potential In addition our data shows, in the RLX group, a positive therapeutic benefits of this molecule in the prevention of correlation between HDL-C and platelet NOS activity cardiovascular disease have recently been evaluated in more confirming that HDL-C decreases platelet function per se than 7000 female osteoporotic participants in the Multiple via an increase in nitric oxide synthase activity as observed Outcomes of Raloxifene Evaluation trial [30]. The authors, by Chen and co-workers [35,36]. Since human platelets are in raloxifene users of this post hoc analysis, did not find unable to synthesize cholesterol, and platelet cholesterol evidence of any early increased risk of cardiovascular events content is regulated by plasma concentration of cholesterol, and highlighted a significant reduction in the risk of this positive correlation suggests that raloxifene may influence, cardiovascular events in the subset of women with increased indirectly, the platelet metabolism via an increased intracellular availability of l-arginine that reduces iNOS expression and At present, the effects of RLX on cardiovascular risk are platelet peroxynitrite levels. Furthermore, the inhibitory effect under evaluation in a large trial in postmenopausal women of HDL-C on platelet reactivity could also be due to the (Raloxifene Use in The Heart trial) with cardiovascular disease inhibition of LDL binding to its platelets' receptor, the and breast cancer as primary endpoints [31].
inhibition of oxidative modification of LDL or the removal To test this hypothesis several authors have studied the of oxidized lipid components from oxidized LDL [37], thereby effects of RLX on NO release from cultured human umbilical preventing these toxic lipids from activating the platelets.
vein endothelial cells and have shown a significant increase In conclusion, our data highlight that RLX regulates platelet of NO production, due to an oestrogen receptor-dependent NO metabolism by a reduction of iNOS, and has beneficial acute stimulation of eNOS enzymatic activity [32]. Others effects on lipid metabolism. In fact, as platelet cholesterol studies have compared the effects of raloxifene therapy and content is regulated by plasma concentration of cholesterol [37], HRT on plasma NO products, endothelin-1 (ET-1) and a reduction in plasma cholesterol levels may increase platelet prostacyclin plasma levels and endothelium-dependent NO production and contribute, thus, to negate the risk of vasodilation in postmenopausal women. These papers cardiovascular events. These mechanisms raise the possibility showed that both treatments influenced, comparatively, that RLX may have the potential to be a cardioprotective agent the endothelial function enhancing the ratio of NO to with the benefit of no increased risk of cancer and other ET-1 [33,34], decreasing prostacyclin levels [33] and improving flow-mediated endothelium-dependent Moreover it is well known that RLX might have a neutral vasodilation [34]. In these trials the improvement of impact on cardiovascular disease, but our study is too short to the endothelium-mediated vasodilation can be substantially demonstrate a realistic decrease in cardiac events. In fact, referred to the reduction of ET-1, an endothelial constrictor a recent study has shown that prothrombotic states may factor, more than to an increase of endothelial NO.
favour occlusive thrombi at sites occupied by atheromatous The present study evaluates, for the first time, in non-obese plaques. Platelet activation has received attention as an postmenopausal women in relation to untreated subjects, the important determinant of arterial thrombogenesis but, metabolic effects of RLX on platelets.
although the evidence is still sparse, the globally available Our findings show that in healthy postmenopausal RLX evidence suggests neutral or beneficial effects for SERMs [38].
users a significantly increased platelet NO activity, an Further work in humans is warranted to enhance our unchanged eNOS expression, a reduced iNOS expression understanding of the mechanisms of the effects of RLX on and a significant decrease in platelet peroxynitrite level platelet metabolism and activity to determine whether the can be achieved. These outcomes, taken together, strongly effects of this drug may contribute to the reduction of indicate that RLX-induced platelet NO production is due cardiovascular related morbidity and mortality in to the reduction of the iNOS isoform. This brings about postmenopausal women. Solid conclusions may be a reduced iNOS-dependent superoxide formation, drawn only after the results of long term clinical studies, resulting in an underproduction of peroxynitrite that that are currently under way, are published.
European Journal of Clinical Investigation Vol 38 123 L. NANETTI ET AL.
15 Rao GH. Measurement of ionised calcium in normal human blood platelets. Anal Biochem 1988;69:400–4.
Institute of Biochemistry, Università Politecnica delle Marche, 16 Camilletti A, Moretti N, Giacchetti G, Faloia E, Martarelli D, Ancona, Italy (L. Nanetti, A. Vignini, F. Raffaelli, L. Mazzanti), Mantero F et al. Decreased nitric oxide levels and increased Unit of Endocrinology, Department of Internal Medicine, calcium content in platelets of hypertensive patients.
Am J Hypertens 2001;14:382–6.
Università Politecnica delle Marche, Ancona, Italy 17 Palmer RMJ, Rees DD, Ashton DS, Moncada S. 1-arginine (A. Camilletti, C. M. Francucci, M. Boscaro).
is the physiological precursor for the formation of nitric oxide Correspondence to: Dr Laura Nanetti, Istituto di Biochimica,
in endothelium-dependent relaxation. Biochem Biophys Res Università Politecnica delle Marche, Via P. Ranieri 65, 60131 Ancona. Tel.: +39 0712204058; Fax: +39 0712204398; 18 Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteine-dye binding. Anal Biochem 1976;72:248–54.
Received 24 May 2007; accepted 26 November 2007 19 Tannous M, Rabini RA, Vignini A, Moretti N, Fumelli P, Zielinski B et al. Evidence for iNOS-dependent peroxynitrite
production in diabetic platelets. Diabetologia 1999;42:539–44.
20 Ertugrul DT, Gürsoy A, Yücel M, Sahin M, Unal AD, Pamuk B 1 Thacker HL. Raloxifene produced both harms and benefits in et al. Effects of raloxifene on platelet functions in patients with postmenopausal women, with no reduction in cardiovascular postmenopausal osteoporosis. Platelets 2006;17:351–3.
disease risk. Evid Based Med 2006;11:178.
21 Lewis SJ. Risk of cardiovascular disease as a woman ages. 2 De Graaf JC, Banga JD, Moncada S, Palmer RMJ, de Groot PG, J Reprod Med 2007;52:147–51.
Sixma JJ. Nitric oxide functions as an inhibitor of platelet 22 Mendelsohn ME, Karas RH. Estrogen and the blood vessel wall. adhesion under flow conditions. Circulation 1992;85:2284–90.
Curr Opin Cardiol 1994;9:619–26.
3 Tousoulis D, Boger RH, Antoniades C, Siasos G, Stefanadi E, 23 Khetawat G, Faraday N, Nealen ML, Vijayan KV, Bolton E, Stefanadis C. Mechanisms of disease: 1-arginine in coronary Noga SJ et al. Human megakaryocytes and platelets contain atherosclerosis – a clinical perspective. Nat Clin Pract Cardiovasc the estrogen receptor beta and androgen receptor (AR): testosterone regulates AR expression. Blood 2000;95:2289–96.
4 Garcia X, Stein F. Nitric oxide. Semin Pediatr Infect Dis 24 Clemens JA, Bennett DR, Black LJ, Jones CD. Effects of a new antiestrogen, keoxifene (LY156758), on growth of 5 Schmidt HHHW, Pollock JS, Nakane M, Forstermann U, carcinogen-induced mammary tumors and on LH and Murad F. Ca2+/calmodulin regulated nitric oxide synthases. prolactin levels. Life Sci 1983;32:2869–75.
Cell Calcium 1992;13:427.
25 Fuchs-Young R, Glasebrook AL, Short LL, Draper MW, Rippy MK, 6 Beckman JS, Koppenol WH. Nitric oxide, superoxide, and Cole HW et al. Raloxifene is a tissue-selective agonist/antagonist peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996;271
that functions through the estrogen receptor. Ann N Y Acad Sci (5 Part 1):C1424–37.
7 Mehta JL, Chen LY, Kone BC, Mehta P, Turner P. Identification 26 Meadows ES, Klein R, Rousculp MD, Smolen L, Ohsfeldt RL, of constitutive and inducible forms of nitric oxide synthase in Johnston JA. Cost-effectiveness of preventative therapies for human platelets. J Lab Clin Med 1995;125:370–7.
postmenopausal women with osteopenia. BMC Womens Health 8 Moro MA, Darley-Usmar VM, Goodwin DA, Read NG, Zamora-Pino R, Feelisch M et al. Paradoxical fate and 27 Walsh BW, Kuller LH, Wild RA, Paul S, Farmer M, biological action of peroxynitrite on human platelets. Lawrence J et al. Effects of raloxifene on serum lipids Proc Natl Acad Sci USA 1994;91:6702–6.
and coagulation factors in healthy postmenopausal women. 9 Busse R, Fleming I. Endothelial dysfunction in atherosclerosis. J Vasc Res 1996;33:181–94.
28 Nickelsen T, Creatsas G, Rechberger T, Depypere H, Erenus M, 10 Kojda G, Harrison DG. Interactions between NO and reactive Quail D et al. Differential effects of raloxifene and continuous oxygen species: pathophysiological importance in atherosclerosis, combined hormone replacement therapy on biochemical hypertension, diabetes and heart failure. Cardiovasc Res markers of cardiovascular risk: results from the Euralox 1 11 Tremoli E, Colli S, Maderna P, Baldassarre D, Di Minno G. 29 Cagnacci A, Paoletti AM, Zanni A, Arangino S, Ibba G, Hypercholesterolaemia and platelets. Semin Thromb Hemost Orrù M et al. Raloxifene does not modify insulin sensitivity and glucose metabolism in postmenopausal women. 12 Writing Group for Women's Health Initiative Investigators Risks and benefits of estrogen plus progestin in healthy 30 Barrett-Connor E, Grady D, Sashegyi A, Anderson PW, Cox DA, postmenopausal women. JAMA 2002;288:321–33.
Hoszowski K et al. Raloxifene and cardiovascular events in 13 Black LJ, Jones CD, Falcone JF. Antagonism of estrogen action osteoporotic postmenopausal women: four-year results from with a new benzothiophene derived antiestrogen. Life Sci the MORE (Multiple Outcomes of Raloxifene Evaluation) randomized trial. JAMA 2002;287:847–57.
14 Figtree GA, Lu Y, Webb CM, Collins P. Raloxifene acutely 31 Wenger NK, Barrett-Connor E, Collins P, Grady D, Kornitzer M, relaxes rabbit coronary arteries in vitro by an estrogen Mosca L et al. Baseline characteristics of participants in the receptor-dependent and nitric oxide-dependent mechanism. Raloxifene Use for The Heart (RUTH) trial. Am J Cardiol 2008 The Authors. Journal Compilation 2008 Blackwell Publishing Ltd RALOXIFENE, ENOS, INOS, AND PLASMA LIPIDS 32 Simoncini T, Genazzani AR. Raloxifene acutely stimulates nitric 35 National Academy of Sciences Committee on Nitrate and oxide release from human endothelial cells via an activation of Alternative Curing Agents in Food. The Health Effects of Nitrate, endothelial nitric oxide synthase. JCEM 2000;85:2966–9.
Nitrite and N-Nitroso Compounds, Part 1. Washington, DC: 33 Saitta A, Altavilla D, Cucinotta D, Morabito N, Frisina N, National Academy Press,1981,pp. 26–32.
Corrado F et al. Randomized, double-blind, placebo-controlled 36 Chen LY, Metha JL. Inhibitory effect of high-density lipoprotein study on effects of raloxifene and hormone replacement on platelet function is mediated by an increase in niric oxide therapy on plasma no concentrations, endothelin-1 levels, synthase activity in platelets. Life Sci 1994;55:1815–21.
and endothelium-dependent vasodilation in postmenopausal 37 Chen LY, Mehta P, Mehta JL. Oxidized LDL decreases 1-arginine women. Arterioscler Thromb Vasc Biol 2001;21:1512–9.
uptake and nitric oxide synthase protein expression in human 34 Christodoulakos G, Panoulis C, Kouskouni E, Chondros C, Dendrinos S, Creatsas G. Effects of estrogen-progestin and 38 Cano A, Hermenegildo C, Oviedo P, Tarín JJ. Selective estrogen raloxifene therapy on nitric oxide, prostacyclin and endothelin-1 receptor modulators and risk for coronary heart disease. synthesis. Gynecol Endocrinol 2002;16:9–17.
European Journal of Clinical Investigation Vol 38 125


Alendronat stada veckotablett 70mg tabl eng

SUMMARY OF PRODUCT CHARACTERISTICS NAME OF THE MEDICINAL PRODUCT Osteomel Once Weekly 70 mg Tablets QUALITATIVE AND QUANTITATIVE COMPOSITION Each tablet contains 70 mg alendronic acid (as sodium alendronate trihydrate). Excipients: each tablet contains 142.64 mg lactose monohydrate. For a full list of excipients, see section 6.1.


Quality Assurance in Higher VET Presented on the examples of the master craftsperson qualification and the WIFI Fachakademie Alexander Petanovitsch Sabine Tritscher-Archan Quality Assurance in Higher Vocational Education and Training Copyright © Vienna, September 2015 The European Commission support for the production of this publication does not constitute an endorsement of the contents which reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein. Editor: Institut für Bildungsforschung der Wirtschaft (ibw) Rainergasse 38, 1050 Wien Austria W Translation: AHA Translation Office, Michael Reiterer Lange Gasse 11/15, A-1080 Vienna, Austria W Project website