Schoenmaker, Email: ln.atca@rekamneohcs.t. T. marker for bone resorption, and procollagen type 1?N propeptide (P1NP), a marker for bone formation) was determined inside a randomized crossover design in 12 healthy volunteers, aged 18C70?years. In addition, we assessed the effect of clonidine on the number of tartrate-resistant acid phosphataseCpositive multinucleated cells (TRAcP+ MNCs) and bone resorption. Results CTx concentrations improved after clonidine treatment compared to the control condition (test. Comparisons of the number of TRAcP+ MNCs and percentage of resorbed bone area over time and between the different culture conditions at day time 21 and day time 28 were made with a KruskalCWallis test, followed by post hoc assessment. All analyses were tested in the 0.05 level of significance. Results In vivo experiment Subject characteristics We enrolled a total of 12 eligible study participants: four males, and five premenopausal and three postmenopausal ladies having a median age of 27 (IQR 32) years. All subjects had calcium, albumin, phosphate, PTH, and alkaline Nalmefene hydrochloride phosphatase concentrations within the research range and normal kidney function. The median 25(OH)D concentration was 50 (IQR 24) nmol/L. None of the participants experienced a (family) history of bone diseases or fractures. Two subjects were current moderate smokers, and two were former smokers. None of them of the study subjects used 2?units?alcohol/day. Three premenopausal ladies used monophasic combined oral contraceptives. All experienced their tablet-free interval during the control day time of the experiment. Two women experienced a regular menstrual cycle and were in the follicular phase during the treatment with clonidine. The additional subjects did not use any medication. Sympathetic nervous system and bone turnover markers Activation of alpha-2-adrenoceptors by clonidine prospects to a reduction in central sympathetic outflow and a resultant decrease in blood pressure, as well as a decrease in catecholamine launch from sympathetic nerves. Plasma normetanephrine, a norepinephrine metabolite, concentrations can be regarded as a marker of sympathetic firmness. As expected, blood pressure (data not demonstrated) and normetanephrine concentrations declined after clonidine treatment (Fig.?1a). Baseline CTx (bone resorption) and P1NP (bone formation) concentrations were not different between the treatment and control days ( em p /em ?=?0.398 and em p /em ?=?0.196, respectively). CTx was higher after clonidine compared to the control condition (time*treatment effect, em p /em ?=?0.035) (Fig.?1b). The effect was most obvious 2?h (11:00) after clonidine administration, with CTx concentrations of 568.5??150.9 versus 474.9??143.1?ng/L in the control condition ( em p /em ?=?0.001). CTx concentrations remained significantly different throughout the remainder of the experiment (13:00, em p /em ?=?0.005, and 15:00, em p /em ?=?0.028). P1NP concentrations were not affected by clonidine ( em p /em ?=?0.520) (Fig.?1c). Open in a separate windows Fig. 1 Normetanephrine (nmol/L) (a); C-terminal cross-linking telopeptides of collagen type I (CTx) (ng/L), a marker for bone resorption (b); and procollagen type 1?N propeptide (P1NP) (g/L), a marker for bone formation (c) within the control (open dots) and treatment (closed dots) time points. Normetanephrine concentrations declined after clonidine treatment (time*treatment effect, em p /em ? ?0.001). CTx was higher after clonidine compared to control (time*treatment effect, em p /em ?=?0.035). P1NP concentrations were not affected by clonidine (time*treatment effect, em p /em ?=?0.520). Data are indicated as mean??SEM. * em p /em ? ?0.05; ** em p /em ? ?0.01; *** em p /em ? ?0.001 In vitro experiment Formation of TRAcP+ MNCs and bone resorption Tartrate-resistant acid phosphataseCpositive (TRAcP+) multinucleated cells (MNCs) were observed after 14?days of culturing (Fig.?2a). The number of TRAcP+ MNCs improved in the course of the experiment, with a significant difference in the number of TRAcP+ MNCs (3C5 nuclei, 5+ nuclei, and total number) between 14 and 28?days of culturing ( em p /em ?=?0.003, em p /em ?=?0.008, and em p /em ?=?0.003). The total quantity of TRAcP+ MNCs improved from 103 (IQR 32) on day time 14 to 649 (IQR 233) TRACP+ MNCs/cm2 on day time 28 of culturing (Fig.?2b). Open in a separate windows Fig. 2 Representative example of tartrate-resistant acid phosphataseCpositive (TRAcP+) multinucleated cell (MNC) (three nuclei or more) ( em arrows /em ) created from CD14+ monocytes isolated from human being buffy coating at day time 21. em Level pub /em ?=?50?m (a). Formation of TRAcP+ MNCs at tradition days 14, 21, and 28. Data are indicated as total number of TRAcP+ MNCs per square centimeter (mean??SEM). ** em p /em ? ?0.01. CLO?=?clonidine (b) Bone resorption pits could be visualized at 14?days of culturing (Fig.?3a). Good formation of TRAcP+ MNCs, there was an increase in bone resorption from 1.1 (IQR 0.7) % to 6.9 (IQR 4.6) % of resorbed bone area between day time 14 and day time 28 of culturing ( em p /em ?=?0.001) (Fig.?3b). Open in a separate windows Fig. 3 Representative example of resorption pits (RP) ( em arrows /em ) stained with Coomassie.Osteoclast-like cells did express mRNA for those alpha-2-adrenergic subtypes, but osteoclast formation and activity were not affected by clonidine. improved after clonidine treatment compared to the control condition (test. Comparisons of the number of TRAcP+ MNCs and percentage of resorbed bone area over time and between the different culture conditions at day time 21 and day time 28 were made with a KruskalCWallis test, followed by post hoc assessment. All analyses were tested in the 0.05 level of significance. Results In vivo experiment Subject characteristics We enrolled a total of 12 eligible study participants: four males, and five premenopausal and three postmenopausal ladies having a median age of 27 (IQR 32) years. All subjects had calcium, albumin, phosphate, PTH, and alkaline phosphatase concentrations within the research range and normal kidney function. The median 25(OH)D concentration was 50 (IQR 24) nmol/L. None of the participants experienced a (family) history of bone diseases or fractures. Two subjects were current moderate smokers, and two were former smokers. None of the study subjects used 2?units?alcohol/day. Three premenopausal ladies used monophasic combined oral contraceptives. All experienced their tablet-free interval during the control day time of the experiment. Two women experienced a regular menstrual cycle and were in the follicular phase during the treatment with clonidine. The additional subjects did not use any medication. Sympathetic nervous system and bone turnover markers Activation of alpha-2-adrenoceptors by clonidine prospects to a reduction in central sympathetic outflow and a resultant decrease in blood pressure, as well as a decrease in catecholamine launch from sympathetic nerves. Plasma normetanephrine, a norepinephrine metabolite, concentrations can be regarded as a marker of sympathetic firmness. As expected, blood pressure (data not demonstrated) and normetanephrine concentrations declined after clonidine treatment (Fig.?1a). Baseline CTx (bone resorption) and P1NP (bone formation) concentrations were not different between the treatment and control days ( em p /em ?=?0.398 and em p /em ?=?0.196, respectively). CTx was higher after clonidine compared to the control condition (time*treatment effect, em p /em ?=?0.035) (Fig.?1b). The effect was most obvious 2?h (11:00) after clonidine administration, with CTx concentrations of 568.5??150.9 versus 474.9??143.1?ng/L in the control condition ( em p /em ?=?0.001). CTx concentrations remained significantly different throughout the remainder of the experiment (13:00, em p /em ?=?0.005, and 15:00, em p /em ?=?0.028). P1NP concentrations were Nalmefene hydrochloride not affected by clonidine ( em p /em ?=?0.520) (Fig.?1c). Open in a separate windows Fig. 1 Normetanephrine (nmol/L) (a); C-terminal cross-linking telopeptides of collagen type I (CTx) (ng/L), a marker for bone resorption (b); and procollagen type 1?N propeptide (P1NP) (g/L), a marker for bone formation (c) within the control (open dots) and treatment (closed dots) time points. Normetanephrine concentrations declined after clonidine treatment (time*treatment effect, em p /em ? ?0.001). CTx was higher after clonidine compared to control (time*treatment effect, em p /em ?=?0.035). P1NP concentrations were not affected by clonidine (time*treatment effect, em p /em ?=?0.520). Data are indicated as mean??SEM. * em p /em ? Nalmefene hydrochloride ?0.05; ** em p /em ? ?0.01; *** em p /em ? ?0.001 In vitro experiment Formation of TRAcP+ MNCs and bone resorption Tartrate-resistant acid phosphataseCpositive (TRAcP+) multinucleated cells (MNCs) were observed after 14?days of culturing (Fig.?2a). The number of TRAcP+ MNCs improved in the course of the experiment, with a significant difference in the number of TRAcP+ MNCs (3C5 nuclei, 5+ nuclei, and total number) between 14 and 28?days of culturing ( em p /em ?=?0.003, em p /em ?=?0.008, and em p /em ?=?0.003). The total quantity of TRAcP+ MNCs improved from 103 (IQR 32) on day time 14 to 649 (IQR 233) TRACP+ MNCs/cm2 on day time 28 of culturing (Fig.?2b). Open in a separate windows Fig. 2 Representative example of tartrate-resistant acid phosphataseCpositive (TRAcP+) multinucleated cell (MNC) (three nuclei or more) ( em arrows /em ) created from CD14+ monocytes isolated from human being buffy coating at day time 21. em Level pub /em ?=?50?m (a). Formation of TRAcP+ MNCs at tradition days 14, 21, and 28. Data are expressed as total number of TRAcP+ MNCs per square centimeter (mean??SEM). ** em p /em ? ?0.01. CLO?=?clonidine (b) Bone resorption pits could be visualized at 14?days of culturing (Fig.?3a). In line with the formation of TRAcP+ MNCs, there was an increase in bone resorption from 1.1 (IQR 0.7) % to 6.9 Rabbit polyclonal to PLEKHG3 (IQR 4.6) % of resorbed bone area between day 14 and day 28 of culturing ( em p /em ?=?0.001) (Fig.?3b). Open in a separate windows Fig. 3 Representative example of resorption pits (RP) ( em arrows /em ) stained with Coomassie brilliant blue at day 21. Scale bar?=?100?m (a). Bone resorption at culture days 14, 21, and 28..
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