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  • 1
    Publication Date: 2016-03-02
    Print ISSN: 0969-7128
    Electronic ISSN: 1476-5462
    Topics: Biology , Medicine
    Published by Springer Nature
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  • 2
    Publication Date: 1982-07-01
    Print ISSN: 0006-2960
    Electronic ISSN: 1520-4995
    Topics: Biology , Chemistry and Pharmacology
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  • 3
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Biochemistry 21 (1982), S. 3508-3513 
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 56 (1995), S. 42-48 
    ISSN: 1432-0827
    Keywords: Phenytoin ; Bone formation ; Osteocalcin ; Alkaline phosphatase ; Osteogenesis ; Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract Long-term use of phenytoin for the treatment of epilepsy has been associated with increased thickness of craniofacial bones. The aim of the present study was to evaluate the possibility that low doses of phenytoin are osteogenic in vivo by measuring the effects of phenytoin administration on serum and bone histomorphometric parameters of bone formation in two rat experiments. In the first experiment, four groups of adult male Sprague-Dawley rats received daily I.P. injections of 0, 5, 50, or 150 mg/kg/day of phenytoin, respectively, for 47 days. Serum alkaline phosphatase (ALP) and osteocalcin were increased by 5 and 50 mg/kg/day phenytoin. The increases in osteocalcin and ALP occurred by day 7 and day 21, respectively. The tibial diaphyseal mineral apposition rate (MAR) at sacrifice (day 48) was significantly increased in rats receiving 5 mg/kg/day phenytoin. At a dose of 150 mg/kg/day, the increase in serum ALP, osteocalcin and MAR was reversed. No significant differences in serum calcium, phosphorus, or 1,25(OH)2D3 levels were seen. In a second experiment, three groups of rats received daily I.P. injection of lower doses of phenytoin (i.e., 0, 1, or 5 mg/kg/day, respectively) for 42 days. Phenytoin also did not affect the growth rate or serum calcium, phosphorus, and 25(OH)D3 levels. Daily injection of 5 mg/kg/day phenytoin significantly increased several measures of bone formation, i.e., serum ALP and osteocalcin, bone ALP, periosteal MAR, and trabecular bone volume. However, rats receiving lower doses of phenytoin (i.e., 1 mg/kg/day) did not show significant increases in the serum bone formation parameters. In contrast, metaphyseal osteoblast surface, osteoblast number, osteoid thickness, surface, and volume were all significantly increased in rats treated in 1 mg/kg/day but not with 5 mg/kg/day phenytoin, suggesting that the tibial diaphysis and metaphysis bone formation parameters might have different dose-dependent responses to phenytoin treatment. Administration of the test doses of phenytoin did not significantly affect the histomorphometric bone resorption parameters. In conclusion, these findings represent the first in vivo evidence that phenytoin at low doses (i.e., between 1 and 5 mg/kg/day) is an osteogenic agent in the rat.
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  • 5
    ISSN: 1432-0827
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract We have recently demonstrated that phenytoin is an osteogenic agent at low doses. The present paper describes observations that a mitogenic dose (i.e., 20 μM in BGJb medium) of fluoride significantly augments the phenytoin-dependent stimulation of normal human bone cell proliferation and alkaline phosphatase (ALP) activity in cell culture. Additionally, the present study was designed to investigate whether fluoride and phenytoin would interact to increase bone formation in rats in vivo. Four groups of weight-matched adult male rats received daily I.P. injection of (1) vehicle (10% DMSO), (2) 5 mg/kg/day phenytoin, (3) 5 mg/kg/day phenytoin and 50 ppm NaF, and (4) 50 ppm NaF and vehicle, respectively, for 36 days. Sodium fluoride (NaF) was delivered in drinking water. Blood samples were drawn weekly and analyzed for serum osteocalcin, ALP, calcium, phosphorus, and 25 (OH)D3. Rats were labeled with fetracycline at day 21 and 30 and histomorphometric analysis was carried out on the tibia at the end of the experiment. Neither agent by itself or together affected the serum calcium, phosphorus, or 25 (OH)D3 levels. All measures of bone formation, i.e., serum osteocalcin level and ALP activity, bone ALP specific activity, mineral apposition rate, bone formation rate, and % bone formation surface, were increased by each agent. Fluoride and phenytoin together produced bigger increases in each parameter than did each agent alone. Trabecular bone volume was increased in the bibial metaphysis by fluoride or phenytoin alone; and when administered together, the two agents produced a greater increase. The combined effect of fluoride and phenytoin on each serum and bone formation parameter appeared to be less than additive. Phenytoin or fluoride alone did not significantly reduce the metaphyseal % resorptive surface. However, treatments with both agents together caused a highly significant reduction in the metaphyseal % resorptive surface. Phenytoin and fluoride together also significantly reduced (by 36%) the mineralization lag time, indicating that these agents did not promote osteomalacia. In summary, fluoride and phenytoin act in concert to stimulate bone formation and increase trabecular bone volume without causing mineralization defects in vivo and thus, may be a potential combination therapy for low bone mass in osteoporosis.
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  • 6
    ISSN: 1432-0827
    Keywords: Hydrochlorothiazide ; Cell Proliferation ; Platelet-derived growth factor ; Mitogen ; Osteoblasts (human)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract Long-term use of hydrochlorothizide (HCTZ), a common diuretic agent for hypertension, has been associated with increased bone density and reduced hip fracture rates in patients. In this study, we sought to examine whether HCTZ has an anabolic effect on the proliferation of human osteoblasts (derived from either vertebrate or rib bone samples) in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell number counting. In medium supplemented with 1% bovine calf serum, HCTZ significantly and reproducibly increased [3H]thymidine incorporation and cell number. The stimulatory effect was dose dependent in a biphasic manner, with the maximal stimulation (approximately 60% above control, P〈0.001) seen at 1 μM HCTZ. In fresh serum-free medium, HCTZ was ineffective as a bone cell mitogen, indicating that the bone cell mitogenic activity of HCTZ required a serum growth factor (GF). HCTZ at doses greater than 10 μM was inhibitory in the presence or the absence of serum, presumably because of the cytotoxic effects. The serum requirement for the bone cell mitogenic activity of HCTZ could be replaced with a conditioned medium (conditioned with normal human osteoblasts for 24 hours), or with a mitogenic dose (1 ng/ml) of PDGF. The GF requirement was specific for PDGF, because other bone cell-derived growth factors (i.e., TGFβ, IGF-I, IGF-II, and bFGF) were unable to replace serum for the bone cell mitogenic activity of HCTZ. In summary, this study shows that (1) HCTZ stimulated the proliferation of normal, untrasformed, human osteoblasts in vitro; (2) the bone cell mitogenic effect of HCTZ required the presence of a serum GF; (3) the serum requirement could be replaced with a bone cell GF in conditioned medium; (4) the GF requirement was specific for PDGF. In conclusion, we have demonstrated for the first time that HCTZ has a direct anabolic effect on human osteoblasts in vitro, and that the mitogenic activity is dependent on the presence of PDGF. Because increased bone cell proliferation is a key determinant of bone formation, these observations raise the interesting possibility that HCTZ could act directly on bone cells to stimulate bone formation in patients.
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  • 7
    ISSN: 1432-0827
    Keywords: Key words: Osteocalcin — Radioimmunoassay — Mouse — Ovariectomy.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. The mouse is frequently used as an animal model to study skeletal mechanisms relevant to humans. Biochemical markers of bone formation and resorption provide one of the key parameters for assessing skeletal metabolism. One biochemical marker that has proven to be useful in the studies of mouse skeletal metabolism is osteocalcin. Assay for osteocalcin is available in the mouse. The present study describes development of an osteocalcin radioimmunoassay (RIA) using a synthetic peptide. Intact osteocalcin purified from mouse bone extracts shows parallel displacement with synthetic peptide. Sensitivity of the RIA was 19 ng/ml. The average (n = 9) intra- and interassay coefficient of variation for two controls was less than 10%; the averaged recoveries were 106%. The osteocalcin concentration measured by peptide RIA shows a high correlation (r = 0.88, n = 117, P 〈 0.0001) with an intact osteocalcin assay. In addition, when the intact assay and peptide assays were applied to evaluate skeletal perturbation, similar results were obtained. Accordingly, osteocalcin levels measured by both intact and peptide-based RIA in 8-week C57BL/6J (n = 8) mice treated with PTH 1-34 were twofold higher compared with the vehicle-treated control group. Further studies of the application of the peptide-based RIA for osteocalcin revealed that osteocalcin levels in 4-week postovariectomized (OVX) C57BL/6N mice (n = 10) were 80% higher than the sham-operated (n = 10) mice receiving vehicle. OVX mice receiving weekly injections of estradiol (400 μg/kg body weight) were 38% lower compared with the OVX group treated with vehicle. In conclusion, the peptide-based RIA has analytical and a discriminative power similar to that of the intact osteocalcin assay but has the advantage that the resources for this assay are much easier to accrue.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 15 (1974), S. 45-54 
    ISSN: 1432-0827
    Keywords: Bone ; Fluoride ; Microhardness ; Mineralization ; Strength
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract Microhardness was measured in sampling sites in the tibial diaphysis of control rats that received less than 1 ppm fluoride in the drinking water, and experimental rats that received 30, 90 and 120 ppm fluoride in the drinking water for 17 days. The latter dose was toxic, as evidenced by a decreased final body weight in this group. By means of tetracycline labelling, it was possible to measure bone hardness in four zones of increasing bone age: I) 3 days, II) 8 days, III) 13 days and IV) 22 days. Zones I through III represented bone formed during fluoride treatment, and Zone IV bone formed before fluoride treatment. In the control group, microhardness increased from Zone I to II, probably because mineral concentration was relatively low in Zone I, and remained constant thereafter. In the 90 and 120 ppm fluoride-treated groups, maximum microhardness was not achieved until Zone III. This delay was probably due to the fact that fluoride in large doses inhibits the rate of mineralization. In the 30 ppm fluoride-treated group, there was no delay in achievement of maximum microhardness; microhardness values in Zones I and III were greater than those in control animals, and microhardness in Zone III was higher than that in Zone IV. These results show that: 1) bone microhardness is increased in bone formed during fluoride treatment in rats given 30 ppm fluoride in the drinking water, 2) toxic doses of fluoride delay, but do not prevent achievement of normal maximum microhardness, and 3) changes in microhardness are seen only in bone formed during fluoride treatment.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 3 (1969), S. 67-73 
    ISSN: 1432-0827
    Keywords: Bones ; Vitamin D ; Enzymes ; Phosphatases ; Hydrolases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé On a fait des recherches sur les conséquences d'un manque de la vitamine D par rapport a la croissance, a la composition ionique du sang et de l'activité encymatique dans l'os chez le rat. La diét contenait 0,6% de calcium et 0,6% de phosphore. Sans apport de vitamine D la croissance était lente, la cendre de la métaphyse diminuait, le serum calcium abaissait le serum phosphore augmentait. L'activité des encymes dans la métaphyse et la diaphyse de la tibia et du femur était la suivante: alkaline phosphatase, acide β-glycerophosphatase, activité protéolytique a pH 8, ta leucin-aminopéptidase et la glutamatdehydrogenase était augmenté dans la métaphyse; la lactatdehydrogenase et l'acide phénylphosphatase était diminué. Des changements comparables étaient decouvertes dans la diaphyse a l'éxception d'une activité de lactatdehydrogenase et de phenylphosphatase non alterée; en outre l'activité proteolytique a pH 5 était augmentée. Le manque de la vitamine D restait sans influence sur l'aspartattrans-aminase et alanintransaminase. L'activité proteolytique a pH 3,7 n'était pas changée. Ces altération encymatiques ne peuvent pas etre provoquées par une malnutrition parce que'elles n'etaient pas observées chez des animales avec une nuriture identique mais a, laquelle on avait ajouté de la vitamin D. Les encymes cataboliques ne manquait pas, la calcémie basse n'est donc pas éxpliquable par une réduction de la résorption osseuse. Les résultats sont plustôt en faveur d'une résorption osseuse très active.
    Abstract: Zusammenfassung Der Effekt von Vitamin D-Mangel auf Wachstum, Serumionen und Enzymaktivität im Knochen wurde bei der Ratte untersucht. Die Diät enthielt 0,6% Calcium und 0,6% Phosphor. Vitamin D-freie Diät hatte verzögertes Wachstum, Abnahme der Knochenasche in der Metaphyse, erniedrigtes Serumcalcium und erhöhtes Serumphosphat zur Folge. Die enzymatische Aktivität in der Metaphyse und Diaphyse von Tibia und Femur veränderte sich: alkalische Phosphatase, saure β-Glycerophosphatase, proteolytische Aktivität bei pH 8, Leucin-Aminopeptidase und Glutamatdehydrogenase waren in Knochenproben der Metaphyse vermehrt, Lactatdehydrogenase und saure Phenylphosphatase vermindert. Ähnliche Veränderungen der Enzymaktivität im Knochen fanden sich in der Diaphyse, mit Ausnahme unveränderter Aktivität von Lactatdehydrogenase und saurer Phenyphosphatase sowie erhöhter proteolytischer Aktivität bei pH 5. Der Vitamin D-Mangel ließ die Aspartat-und Alanin-Transaminasen sowie die proteolytische Aktivität bei pH 3,7 unverändert. Die Enzymveränderungen sind nicht durch geringere Nahrungsaufnahme bedingt, weil sie bei Tieren mit identischer Ernährung, aber ohne Vitamin D-Entzug, nicht beobachtet wurden. Ein Mangel an Abbaufermenten, welcher Hemmung der Knochenresorption bewirken und für den Abfall des Serumcalciums verantwortlich sein könnte, ließ sich nicht finden. Die Resultate sprechen eher für vermehrte Knochenresorption.
    Notes: Abstract The effect of vitamin D deficiency on growth, serum ions and bone enzymatic activity has been studied in rats. The diet contained 0.6% calcium and 0.6% phosphorus. Omission of vitamin D from the diet resulted in decreased growth, decreased metaphyseal bone ash, decreased serum calcium and increased serum phosphorus. Changes in enzymatic activity in metaphyseal and diaphyseal bone from the tibia and femur also occurred. Alkaline phosphatase, acid β-glycerophosphatase, proteolytic activity at pH 8.0, leucine aminopeptidase, and glutamate dehydrogenase were increased in metaphyseal bone samples. Lactate dehydrogenase and acid phenylphosphatase were decreased. Similar changes in bone enzymatic activity occurred in the diaphysis except that the lactate dehydrogenase and acid phenylphosphatase activities were not decreased and proteolytic activity at pH 5.6 was increased. The vitamin D deficiency did not affect aspartate or alanine transaminase or proteolytic activity at pH 3.7. These enzyme changes were not due to decreased food consumption because they did not occur in pair-fed animals. There was no lack of degradative enzymes that would impair bone resorption and lead to a fall in serum calcium. Rather the results were more consistent with an increase in bone resorption.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 3 (1969), S. 55-66 
    ISSN: 1432-0827
    Keywords: Bones ; Bone marrow ; Enzymes ; Phosphatases ; Hydrolases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé Les activités de différentes encymes du fémur et de la tibia du rat étaient étudiées. Pour mesurer acide et alkaline phosphatase, lactatdehydrogenase, activité protéolytique, leucinaminopéptidase, glycylglycindipéptidase, glutamatdehydrogenase, aspartate transaminase et alanintransaminase, une méthode quantitative était developpé. Quelques réactions characteristiques étaient examinées das n des homogénates de la métaphyse. La distribution des encymes dans la métaphyse, diaphyse et dans la moelee osseuse était étudié. L'activité la plus grande per mg DNA était observé dans la métaphyse, celle la plus basse dans la moelle osseuse. En générale les activités étaient 40–70% abaissées dans la diaphyse comparé avec la métaphyse. L'activité encymatique de la métaphyse n'était pas différente l'une de l'autre chez les rats agés de 29 et 49 jours mais elle diminuait chez celles de 82 jours. L'activité protéolytique a pH 8 ne correspondait pas avec les autres encymes: elle était plus haut dans la moelle osseuse que dans l'os et augmentait visiblement avec l'age.
    Abstract: Zusammenfassung Verschiedene Enzymaktivitäten in Femur und Tibia der Ratte wurden untersucht. Es wurden quantitative Prüfmethoden für die Bestimmung von saurer und alkalischer Phosphatase, Lactatdehydrogenase, proteolytischer Aktivität, Leucinaminopeptidase, Glycylglycin-Dipeptidase, Glutamatdehydrogenase, Aspartat-Transaminase und Alanin-Transaminase entwickelt. Einige Eigenschaften dieser Enzyme wurden in Metaphysenhomogenaten untersucht. Die Verteilung der Enzymaktiväten in Metaphyse, Diaphyse und Knochenmark wurde bestimmt. Die Aktivität pro mg DNA war am höchsten in der Metaphyse, am niedrigsten im Knochenmark. Allgemein waren die Aktivitäten 40–70% tiefer in der Diaphyse als in der Metaphyse. Die metaphysäre enzymatische Aktivität war dieselbe bei 29 und 49 Tage alten Ratten, hingegen tiefer bei 82 Tage alten Ratten. Die proteolytische Aktivität bei pH 8 war von anderen untersuchten Aktivitäten verschieden; sic war höher im Knochenmark als im Knochen und zeigte deutliche Zunahme mit dem Alter.
    Notes: Abstract A study was made of several enzymatic activities in rat femur and tibia. Quantitative assays were developed for the determination of acid and alkaline phosphatase, lactate dehydrogenase, proteolytic activity, leucine aminopeptidase, glycylglycine dipeptidase, glutamate dehydrogenase, aspartate transaminase, and alanine transaminase. Some properties of these enzymes were studied using metaphyseal homogenates. The distribution of activity in the metaphysis, diaphysis, and marrow was determined. The activity per mg of DNA was highest in the metaphysis and lowest in marrow. The activities in general were 40–70% lower in the diaphysis than in the metaphysis. Metaphyseal enzymatic activity was the same in 29- and 49-day-old rats but lower in 82-day-old rats. Proteolytic activity at pH 8.0 differed from the other activities studied being higher in marrow than in bone and increasing markedly with age.
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