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  • Calcification
  • Calcitonin
  • Springer  (65)
  • American Chemical Society
  • Institute of Electrical and Electronics Engineers (IEEE)
  • Springer Science + Business Media
  • 1980-1984  (43)
  • 1965-1969  (22)
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Year
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 33 (1981), S. 417-424 
    ISSN: 1432-0827
    Keywords: Calcitonin ; Calcium conservation ; Calcium storage ; Bone ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary The postulate tested by these experiments is that calcitonin directs a portion of the calcium absorbed from food into temporary storage at surfaces of bone. This study utilized young adult rats in which the parathyroid glands had been autotransplanted. Many rats were also thyroidectomized (TX). All were trained to a 0900 h feeding schedule with predetermined calcium content of food. Calcitonin was injected only during the first 4 h postprandially to some of the TX animals. Urine calcium was monitored and, after sacrifice, changes in the tibia were recorded. The following results were obtained: (a) In thyroid-intact (TI) rats, renal calcium excretion was reduced if the daily intake of calcium was less than 90 mg. In TX rats calcium excretion rose each day during the time of intestinal absorption of calcium. Calcitonin injection to TX rats reduced urinary calcium content on the day it was injected. However, on the following 2 days, renal calcium excretion rose to twice that of TX controls. (b) Electron micrographs of bone tissue from TI rats and TX rats injected with calcitonin showed greater pyroantimonate reaction within bone fluid than did tissue from TX controls. Similarly, when prepared by an anhydrous procedure, bone surfaces of tibia taken from rats with endogenous or exogenous calcitonin contained dense material accumulation (following a calcium-containing meal) not present in bones from TX rats. (c) When tibia shaft fragments of rats sacrificed 4 h after consuming a calcium-containing meal were washed in acidic saline, more calcium accumulated during the first 15 min in the media containing bones from TI than from TX rats. The final equilibration level between media and bone fragments was not affected by the calcitonin state of the rat. These experiments demonstrate both physiological and bone morphological differences between TI and TX rats which can be negated by postprandial calcitonin injection to TX animals. They support the postulate that the secretion of calcitonin postprandially aids in the conservation and storage of ingested calcium.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 2 (1968), S. 296-298 
    ISSN: 1432-0827
    Keywords: Calcium ; Lipid ; Bacteria ; Calcification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé On a fait ce travail pour determiner si le facteur responsable pour la liaison de calcium par un calcifableBacterionema matruchotii est dans la fraction lipide de la cellule. Des cellules congelees et sechees ont ete extraites par le chloroform-methanol. La fraction de chloroform-methanol, les cellules extraites et les cellules non traitees ont ete examinees pour la liaison de calcium. La fraction du chloroform-methanol et les cellules non traitees avaient la liaison de calcium. Les cellules extraites n'en avaient pas.
    Abstract: Zusammenfassung Diese Arbeit wurde durchgeführt um festzustellen, ob sich der Faktor für die Calcium-bindung, durch das calcifizierendeBacterionema matruchotii, in der Lipoidfraktion befindet. Die lyophiilisierten Zellen wurden mit Chloroform-Methanol extrahiert. Die Chloroform-Methanol-Fraktion, die extrahierten Zellen, sowie die nicht behandelten Zellen wurden auf eine Calciumbindung hin untersucht. Die Chloroform-Methanol-Fraktion und die nicht behandelten Zellen demonstrierten eine Calciumbindung. Die extrahierten Zellen hingegen nicht.
    Notes: Abstract This work was done to determine whether the factor responsible for calcium binding by a calcifiableBacterionema matruchotii is in the lipid fraction of the cell. Freeze-dried cells were extracted with chloroform-methanol. The chloroform-methanol fraction, the extracted cells and untreated cells were examined for calcium binding. The chloroform-methanol fraction and the untreated cells bound calcium. The extracted cells did not.
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  • 3
    ISSN: 1432-0827
    Keywords: Calcitonin ; Cyclic GMP ; Kidney
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary In order to evaluate whether or not the action of salmon calcitonin (sCT) at the kidney level could be mediated through specific receptors for the hormone, we have studied the effects of sCT infusions on urinary excretion of cyclic nucleotides in humans. Parallel in vitro studies have been conducted by evaluating the effects of sCT on cyclic nucleotide levels in primary cultures of cortical and medullary human kidney cells. In vivo experiments showed that sCT induced an increase in cGMP in human urine, which was rapid and short-lasting, being superimposable on the increase of urinary excretion of calcium and magnesium. The increase of inorganic phosphate urinary excretion was delayed and appeared to parallel that of urinary cAMP. On the other hand, our in vitro experiments showed that sCT stimulated the guanylate cyclase—cGMP system of human kidney cortical cells at nanomolar concentrations, while higher concentrations of the hormone were required to activate the adenylate cyclase—cAMP system. In addition, sCT was not able to significantly modify the cellular levels of either nucleotide in human kidney medullary cells. Present data demonstrated a direct effect of sCT on human kidney cortical cGMP production, while the efficacy of sCT on the kidney cortex adenylate cyclase—cAMP system appears to be delayed and/or reduced.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 32 (1980), S. 221-228 
    ISSN: 1432-0827
    Keywords: Medullary bone ; Calcification ; Low-calcium diet ; Parathyroid hormone ; Estrogens
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary Medullary bone of birds maintained on a low-calcium diet represents a good model to study modifications of matrix composition in calcified tissue undergoing intense formation and resorption. The composition of the bone matrix during the low-calcium diet has been analyzed by both chemical and histological techniques. Sixty White Leghorn pullets 1 year old were used for the experiment. Fifteen birds served as controls and were killed on day zero; the remaining birds were placed on a calcium-deficient diet (0.13% calcium) and sacrificed after 4, 7, and 12 days of treatment in groups of 15. Serum levels of calcium, PTH, and estrogens were also measured. Chemical analysis of the samples were made for total nitrogen, hydroxyproline, hexosamine, hexoses, calcium, and phosphorus. Collagen and proteoglycans of the matrix of medullary bone of the egg-laying hens were found to be affected by the low-calcium diet. They either increased or decreased during the experiment but never in parallel. The increment of serum PTH is considered responsible for the variations in the amount of collagen. The effects of this hormone are magnified by the fall of serum estrogens as shown also by variations in the amounts of noncollagenous protein. In the late phase of the diet the matrix is represented by poorly calcified osteoid tissue rich in noncollagenous protein, i.e., proteoglycans and glycoproteins.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 35 (1983), S. 502-507 
    ISSN: 1432-0827
    Keywords: Estradiol ; Hypercalcemia ; Calcitonin ; Oopherectomy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary To investigate interactions between estrogens and calcitonin (CT), we have studied the effect of oophorectomy and estrogen replacement on plasma CT in female rats in response to an acute intravenous calcium stimulus and to hypercalcemia induced by dietary phosphorus deprivation. Plasma CT 5 min after an intravenous calcium stimulus (10 mg Ca/kg) was 176±11 pg/ml (mean ± SEM) in rats subjected to oophorectomy compared with 244±15 pg/ml in sham-operated control rats (P〈.001). Administration of estradiol benzoate (10 µg/kg/day) for 14 days restored the stimulated plasma CT concentration to 221±15 pg/ml (P〈.02 compared with oophorectomy). On regression of plasma CT against calcium, including both basal and stimulated values, the slopes (± standard error of estimate) were 23.7±5.0 pg CT/mg Ca for controls, 16.1±4.0 after oophorectomy, and 26.0 ± 3.2 after estrogens. Rats placed on a low phosphorus diet demonstrated a fall in plasma phosphorus, a rise in plasma calcium, a decrease in plasma parathyroid hormone (PTH), and a rise in plasma CT after phosphorus depletion. In male rats, plasma CT was maximal on day 2 of phosphorus depletion (109±15 pg/ml vs 47±4 pg/ml for controls,P〈.001), and continued phosphorus deprivation was associated with a significantly decreased plasma CT response to an intravenous calcium stimulus (mean 167±9 vs 284±21 pg/ml,P〈.001). There was an inconsistent effect of oophorectomy in lowering CT levels on day 2 of phosphorus depletion and no effect on day 15 (94±19 pg/ml vs 82±9 pg/ml). We conclude that estrogens increase the plasma CT response to an acute intravenous calcium stimulus. We examined the effect of phosphorus depletion on CT as a model for endogenous hypercalcemia. Phosphorus deprivation was found to be a potent stimulus to plasma CT, but we could not consistently demonstrate an effect of endogenous estrogens on plasma CT under these conditions (estrogen-hypophosphatemia-calcitonin).
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 35 (1983), S. 566-570 
    ISSN: 1432-0827
    Keywords: Osteoclast ; Motility ; Calcitonin ; Prostacyclin ; Cyclic AMP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary We separated osteoclasts from bone and observed the effect of several known and potential mediators of the control of bone resorption on their cytoplasmic motility. We already found that calcitonin (CT), a hormone that inhibits bone resorption, regularly causes complete inhibition of cytoplasmic motility, specific for osteoclasts, through a trypsin-sensitive membrane receptor [1]. We report here that prostaglandin I2 (PGI2) and dibutyryl cyclic AMP induce an identical change in osteoclastic behavior. We found that theophylline, which inhibits intracellular cyclic AMP degradation, and which itself had no effect on osteoclastic motility, potentiated the cytoplasmic inhibition casued by CT, PGI2, and cyclic AMP. This suggests that PGI2 and CT cause cytoplasmic quiescence by increasing the intracellular level of cyclic AMP, a view compatible with the known ability of CT to increase cyclic AMP in bone [2]. Parathyroid hormone (PTH), PGE2, and 1,25 dihydroxycholecalciferol (1,25 (OH)2D3), hormones known to stimulate osteoclasts, did not stimulate the activity of either active or quiescent isolated osteoclasts. The undoubted ability of these hormones to stimulate osteoclastic activityin vivo may therefore be mediated through a primary hormonal interaction with another cell type.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 35 (1983), S. 723-727 
    ISSN: 1432-0827
    Keywords: Calcification ; Scleroderma ; CRST syndrome ; High resolution TEM ; Microanalysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary This paper reports the study of a subcutaneous heterotopic calcification from a patient with Thibierge-Weissenbach's syndrome, a type of creeping scleroderma included in the CRST syndrome. These local deposits, whose origin is still unknown, are commonly considered to be a classic apatite phase. Using SEM, high resolution TEM, electron diffraction, infrared spectrometry, and SEM and TEM microanalysis, it is demonstrated that this material is highly heterogeneous and appears in a nonstoichiometric, carbonated, calcium ion-deficient apatitic solid phase. Our study shows the co-existence of dense globules presenting an ill-organized, more or less amorphous phase (ACP) or microcrystalline (OCP, β tricalcium phosphate), with scattered apatite crystals, and of interglobular apatite crystals with a good cristallinity.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 4 (1969), S. 260-268 
    ISSN: 1432-0827
    Keywords: Cartilage ; Histochemistry ; Staining ; Protein ; Polysaccharide ; Calcification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé Des coupes de cartilage épiphysaire frais de jeunes rats, effectuées à la main, sont colorées à pH=4,5 dans des solutions à 0,01% de divers colorants cationiques, appartenant aux groupes de la thiazine, oxazine, azine, triphénylméthane, acridine, et phthallocyanine. Les granules intracellulaires métachromatiques, mises en évidence antérieurement par le bleu de toluidine, sont également identifiées à l'aide de l'azur A, le bleu de méthylène et le bleu de crésyl. Les granules se colorent moins bien à la thionine, le rouge neutre, la safranine O, le bleu de toluylène et l'acridine orange. Dans les conditions utilisées, la matrice de la zone de réserve et la matrice de la zone hypertrophique inférieure (en voie de calcification) se colorent, alors que les matrices des zones prolifératives et hypertrophiques supérieures ne prennent pas les colorants. La gallocyanine, le violet cristal, la fuchsine basique, l'azocarmin B, le bleu de gallamine et la bleu alcian ne se colorent pas ou donnent des réactions colorées différentes de celles décrites ci-dessus. Il semble que le pK et le poids moléculaire des colorants jouent un rôle important, mais ils ne paraissent pas être les seuls facteurs intervenant dans la coloration des granules. Un changement, lié à la calcification, semble intervenir au niveau du matériel métachromatique (probablement des polysaccharides protéiques), aussi bien dans la matrice que les cellules cartilagineuses épiphysaires.
    Abstract: Zusammenfassung Handpräparierte Schnitte von frischem Epiphysenknorpel junger Ratten wurden bei einem pH von 4,5 in 0,01% igen Lösungen verschiedener kationischer Farbstoffe folgender Klassen gefärbt: Thiazin, Oxazin, Azin, Triphenylmethan, Acridin und Phthalocyanin. Die intracellulären β-und γ-metachromatischen Granula, erstmals mit Toluidinblau im frischen Gewebe nachgewiesen, konnten auch gut mit Azur A, Methylenblau und Brillantkresylblau dargestellt werden. Die Granula konnten ebenfalls, aber weniger gut, mit Thionin, Neutralrot, Safranin D, Toluylenblau und Acridinorange gefärbt werden. Unter diesen Färbungsbedingungen werden die inaktive Matrixzone und die untere hypertrophische (verkalkende) Matrixzone angefärbt, während die proliferative und die obere hypertrophische Matrixzone sich nicht färben. Gallocyanin, Kristallviolett, basisches Fuchsin, Azokarmin B, Gallaminblau und Alzianblau färbten entweder gar nicht, oder gaben ein anderes als das obenbeschriebene Färbemuster. Es wird vorgeschlagen, daß das pK und das Molekulargewicht der Farbstoffe wichtig aber nicht unbedingt die einzigen Faktoren sind, die die Färbung der Granula bestimmen. Die Resultate zeigen, daß eine Veränderung im metachromatischen Material (vermutlich Proteinpolysaccharide) vorliegt, und zwar sowohl in der Matrix als in den Zellen des Epiphysenknorpels; diese Veränderung scheint im Zusammenhang mit der Verkalkung zu stehen.
    Notes: Abstract Hand-cut sections of fresh epiphyseal cartilage from young rats were stained at pH 4.5 in 0.01% solutions of various cationic dyes of the thiazine, oxazine, azine, triphenylmethane, acridine, and phthallocyanin classes. The intracellular β-and γ-metachromatic granules, previously demonstrated in fresh tissues with toluidine blue, were also demonstrated well with azure A, methylene blue, and brilliant cresyl blue. The granules were also demonstrated, but not as well, by thionin, neutral red, safranin O, toluylene blue, and acridine orange. Under the conditions of staining, the reserve zone matrix and the lower hypertrophic (calcifying) zone matrix stained, whereas the proliferative and upper hypertrophic zone matrix did not stain. Gallocyanin, crystal violet, basic fuchsin, azocarmine B, gallamine blue, and alcian blue either did not stain, or gave a different pattern of staining from that described above. It is suggested that the pK and molecular weight of the dyes are important, but not necessarily the only factors in determining the staining of the granules. The results indicate that there is a change in the metachromatic material (presumably proteinpolysaccharide) in both the matrix and cells of epiphyseal cartilage, which appears to be related to calcification.
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  • 9
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    Electronic Resource
    Springer
    Calcified tissue international 34 (1982), S. 470-473 
    ISSN: 1432-0827
    Keywords: Calcitonin ; Premature ; Osteopenia ; Hypocalcemia ; Parathyroid hormone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary Twenty-eight premature infants of mean gestation 30.9±2.5 weeks and mean birth weight 1175±206 g had repeated serum calcitonin concentrations determined over the first 12 weeks of life. Serum calcitonin concentrations slowly fell but remained elevated even at 12 weeks of age [normal adult=71±48, 1 week (N=15)=327±167, 3 week (N=23)=270±129, 6 week (N=16)=249±154, 9 week (N=13)=214±108, 12 week (N=12)=174±11]. Throughout this period, serum total calcium was normal or low (8.4±.8–9.3±1.0). Serum phosphorus was normal or low (6.0±1.4–6.5±1.0), and serum magnesium was normal (1.7±0.24–1.8±0.34). The reason for the sustained elevation of serum calcitonin in these very small, sick, premature infants in unclear.
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  • 10
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    Springer
    Calcified tissue international 4 (1969), S. 20-38 
    ISSN: 1432-0827
    Keywords: Calcification ; Epiphyseal Cartilage ; Bone ; Electrolytes ; Organic matrices
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Description / Table of Contents: Résumé Un procédé de dissection a été mis au point pour permettre l'analyse zonale du cartilage de l'épiphyse des os de la jambe d'un foetus bovin. Des échantillons de tissu complet et lavé venant des différentes zones ont été analysés pour déterminer leur contenu en électrolyte et en constituants organiques, ainsi que pour leur densité, cendres et humidité. Les résultats ont montré que lorsque la quantité de cendres et la densité augmentaient, l'eau contenu dans le tissu diminuait. Les quantités de cendres dans les zones de cartilage en voie de calcification étaient plus grandes qu'il avait été. Quand elles étaient exprimées comme un pourcentage du poids sec, elles étaient les plus importantes dans le cartilage lavé calcifié que dans le autre zones. Au début de la minéralisation du cartilage, la quantité de Na (m moles/l de tissu frais) diminuait tandis que celles du Ca et du P inorganique augmentaient. Les niveaux de Mg augmentaient pendant que la calcification se poursuivait, mais seulement à une faction du taux du Ca et du P. Les rapports Ca/P inorganique étaient les plus grands dans le cartilage au repos (Cartilage non-différentié hyalin), suggérant un lien initiale entre Ca et les chrondromucoprotéines. Cependant, au début de la calcification, pendant la prolifération du cartilage les rapports Ca/P étaient beaucoup plus petits (ca. 1.50) mais augmentaient graduellement avec l'advancement de la minéralisation. Des changements importants survenaient dans la composition de la phase organique, pendant la calcification endochondrale. Comme il a été déterminé par l'analyse de l'hydroxyproline la quantité de collagéne diminuait progressivement pendant la calcification du cartilage, mais augmentait rapidement pendant la formation d'os. Comme il a été déterminé par l'analyse de l'héxosamine et du sulfute les chrondromucoprotéines étaient aux niveaux les plus éléves pendant la prolifération du cartilage et diminuaient constamment au cours de la calcification. Cependant, bien que la calcification était déja très avancée dans le cartilage hypertrophique, de grandes quantites de mucopolysaccharides étaient encore présentes. Les rapports sulfure/hhéxosamine montraient un léger déclin pendant les premiéres étapes de la calcification, mais augmentaient beaucoup pendant le cours de la minéralisation. Les quantités d'acide sialique étaient plus grandes dans le cartilage de l'épiphyse que dans le cartilage au repos ou dans l'os. Les lipides augmentaient rapidement pendant la calcification du cartilage, mais étaient très réduites dans l'os complètement formé. La signification de ces résultats est discutée.
    Abstract: Zusammenfassung Eine Seziermethode, die eine Schichten-Analyse der Beinepiphysenplatte von Rinderfeten erlaubt, wurde entwickelt. Proben vor und nach Waschen des Gewebes der verschiedenen Schichten werden untersucht in bezug auf Elektrolyte und organische Bestandteile, als auch in bezug auf Dichte, Aschengehalt und Feuchtigkeit. Die Resultate zeigten eine Zunahme des Aschengehaltes und der Dichte, während der Wassergehalt abnahm. Unerwartet hoch waren die Aschenwerte im in Verkalkung begriffenen Knorpel. Ausgedrückt in Prozent Trockengewicht, ergab gewaschener, verkalkter Knorpel den höchsten Wert aller Zonen. In den Frühstadien der Knorpelmineralisation nahm der Natriumgehalt (m Mol/l Frischgewebe) ab, während Ca und anorganischer P zunahmen. Mit fortschreitender Verkalkung erhöhte sich auch der Magnesium-Spiegel, allerdings nur zu einem Bruchteil des Ausmaßes, in welchem Ca und P zunahmen. Die höchsten Ca/P anorg. Verhältnisse wurden im Ruheknorpel (undifferenzierter hyaliner Knorpel) gefunden, was auf eine initiale Bindung von Ca durch Chondromucoproteine hinweist. Die Ca/P-Verhältnisse proliferierenden Knorpels waren jedoch bei Verkalkungsbeginn viel tiefer (ca. 1.50). Diese nahmen allerdings mit fortschreitender Mineralisierung stetig zu. In der endochondralen Verkalkungsphase fanden markante Veränderungen in der Zusammensetzung des organischen Anteils statt. Basierend auf der Hydroxyprolinanalyse nahm der Collagengehalt in der knorpeligen Verkalkungsperiode fortschreitend ab, während er jedoch bei der Knochenbildung rasch zunahm. Die an Hand von Hexosamin- und Schwefelanalysen bestimmten Chondromucoproteingehalte ergaben Höchstwerte im proliferierenden Knorpel und fielen stetig ab mit zunehmender Verkalkung. Trotz der im hypertrophischen Knorpel schon weit fortgeschrittenen Verkalkung waren immer noch große Mengen an Mucopolysacchariden vorhanden. Die Schwefel/Hexosamin-Verhältnisse zeigten eine minimale Abnahme in den frühen Verkalkungsphasen, nahmen jedoch markant zu bei fortschreitender Mineralisation. Der Sialinsäurespiegel war im Epiphysenknorpel, verglichen mit demjenigen des Ruheknorpels oder Knochens, erhöht. In der knorpeligen Verkalkungsphase nahmen die Lipide rasch zu, während jedoch die Werte des vollständig ausgebildeten Knochens stark vermindert waren. Die Bedeutung dieser Ergebnisse wird besprochen.
    Notes: Abstract A dissection procedure has been devised to permit zonal analysis of the epiphyseal plate of fetal calf leg bones. Samples of whole and washed tissue from the various zones were analyzed for their content of electrolyte and organic constituents, as well as for density, ash and moisture. Results showed that as ash content and density increased, water content decreased. Ash levels in calcifying cartilage zones were unexpectedly high. When expressed as a percentage of dry weight, washed calcified cartilage had the highest content of any zone. In the early stages of the mineralization of cartilage, Na content (mmoles/l of fresh tissue) decreased as Ca and inorganic P increased. Magnesium levels increased as calcification proceeded, but only at a fraction of the rate of Ca and P. Ratios of Ca/inorganic P were highest in resting cartilage (non-differentiated hyaline cartilage), suggesting an initial binding of Ca to chondromucoproteins. However, at the onset of calcification in proliferating cartilage, Ca/P ratios were much lower (ca. 1.50), but gradually increased with advancing mineralization. Marked changes occurred in the composition of the organic phase during endochondral calcification. As determined by hydroxyproline analysis, collagen content progressively decreased during cartilaginous calcification, but increased rapidly during bone formation. As determined by hexosamine and sulfur analysis, chondromucoproteins were at highest levels in proliferating cartilage and decreased steadily as calcification increased. However, although calcification was already well advanced in hypertrophic cartilage, large amounts of mucopolysaccharide still were present. Sulfur/hexosamine ratios showed a slight decline during the early stages of calcification, but increased markedly with further mineralization. Sialic acid levels were elevated in epiphyseal cartilage over those in resting cartilage or bone. Lipids increased rapidly during cartilaginous calcification, but were greatly reduced in fully-formed bone. The significance of these findings is discussed.
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