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PubMed1992

Kinetics of beta-glycerophosphate-induced endochondral mineralization in vitro. Calcium accumulation, alkaline phosphatase activity, and effects of levamisole.

Isolated mesenchymal limb bud cells from day-12 mouse embryos grown at high density in organoid culture at the medium/air interphase differentiate into chondrocytes and form cartilage nodules. Upon addition of beta-glycerophosphate (beta-GP), cartilage undergoes endochondral mineralization. This beta-GP-induced mineralization was investigated by measuring the calcium content in the cultures and the activity of alkaline phosphatase (AP) in the cell mass and the medium. Calcium incorporation depended on the amount of beta-GP added. After continuous treatment, mineralization began on day 8 of the culture period and increased linearly until day 15. In long-term cultures, periodical treatment for 6 days caused an increase in mineralization the older the cultures were, but the slope of increase was proportionately less steep. Treatment at the latest period on days 19-24 resulted in a markedly reduced mineralization. After short-term treatment (48 hours), mineralization increased also the older the cultures were and proceeded during further cultivation in beta-GP-free medium. This kinetic behavior indicates a dependency of mineralization on cartilage maturation in this in vitro system. AP activity increased enormously and nearly logarithmically in the cell mass in beta-GP-free medium, whereas beta-GP treatment inhibited this drastic increase. In the medium, considerable activities of AP were also measurable from day 10 onward. It increased in beta-GP-free medium up to day 14, but was diminished after mineralization had been induced. Levamisole inhibited AP activity dose dependently when added directly to the enzyme-containing medium (100% inhibition at 10(-3) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1992

Mechanism of action of beta-glycerophosphate on bone cell mineralization.

Experiments were performed to determine whether beta-glycerophosphate (beta-GP) promoted mineralization in vitro by modulating bone cell metabolic activity and/or serving as a local source of inorganic phosphate ions (Pi). Using MC3T3-E1, ROS 17/2.8, and chick osteoblast-like cells in the presence of beta-GP or Pi, we examined mineral formation, lactate generation, alkaline phosphatase (AP) activity, and protein and phospholipid synthesis. Neither beta-GP nor Pi modulated any of the major biosynthetic activities of the bone cells. Thus, we found no change in the levels of phospholipids, and the total protein concentration remained constant. Measurement of lactate synthesis showed that beta-GP did not effect the rate of anaerobic glycolysis. Evaluation of medium Pi levels clearly indicated that beta-GP was hydrolyzed by bone cells; within 24 hours, almost 80% of 10 mM beta-GP was hydrolyzed. It is likely that this local increase in medium Pi concentration promoted rapid mineral deposition. Chemical, energy dispersive X-ray, and Fourier transform infrared analysis of the mineral formed in the presence of beta-GP showed that it was nonapatitic; moreover, mineral particles were also seen in the culture medium itself. Experiments performed with a cell-free system indicated that mineral particles formed spontaneously in the presence of AP and beta-GP and were deposited into a collagen matrix. We conclude that medium supplementation with beta-GP or Pi should not exceed 2 mM. If this value is exceeded, then there will be nonphysiological mineral deposition in the bone cell culture.

Absorptiometry, Photon↗

PubMed1992

Induction of normal and dystrophic mineralization by glycerophosphates in long-term bone organ culture.

The effectiveness of Na-beta-, and Ca-glycerophosphates (GPs) in inducing mineralization was tested during long-term organ culture of femurs from 14-day-old chick embryos. When bones were incubated with Na-GP, a 66% rise in inorganic phosphate level was measured in the medium, supporting the notion that provision of a substrate for alkaline phosphatase (ALP) increased available phosphate. On the other hand, if the concentrations of Ca2+ were raised, available inorganic phosphate was decreased. Similarly, increases in inorganic phosphate decreased available calcium. Both GPs induced mineralization in bone and cartilage, but more matrix was mineralized with Ca-GP. However, the induction of mineralization by GPs was accompanied by dystrophic calcification, reduction of matrix formation and ALP activity, and increased release of lactate dehydrogenase into the culture medium. The new osteoid, which formed during culture, mineralized in the absence of GPs without the above adverse effects provided the culture period was longer than 15 days. The described organ culture system therefore facilitates studies of the mechanism of bone mineralization without the disadvantages of GP addition.

Alkaline Phosphatase↗

PubMed1990

Matrix vesicles isolated from apical pulp of rat incisors: crystal formation in low Ca x Pi ion-product medium containing beta-glycerophosphate.

The ultrastructure of crystal formation in vitro associated with extracellular membrane-bound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-beta-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultra-centrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30-200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca) x inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.

Alkaline Phosphatase↗

PubMed2000

Development of mineralized nodules in fetal rat mandibular osteogenic precursor cells: requirement for dexamethasone but not for beta-glycerophosphate.

We have reported that a cell population obtained from fetal rat mandible with neutral protease (Pro I) has a unique differentiation sequence in which the elevation of alkaline phosphatase (ALPase), calcium accumulation, and collagen synthesis occurs simultaneously. In this report, we further characterized Pro I-released population of cells by studying the effect of dexamethasone (Dex) or beta-glycerophosphate (beta-GP) on the formation of bone nodules. The formation of bone nodules in Pro I-released population of cells (ProIRPC) was augmented by the addition of Dex (10(-7) M) from days 3 to 14, suggesting that Pro IRPC contained osteoprogenitor (OP) cells. A 24-hour pulse treatment of ProIRPC released population of cells with Dex on days 9 and 12 resulted in an increase in the number of nodules but treatment on days 3, 6, or 15 did not. The number of bone nodules formed in Pro IRPC pulse treated with Dex on day 9 was comparable with that in Pro IRPC treated with Dex from days 3 to 14. Dex caused an earlier elevation of ALPase, in which maximal expression was observed on day 10. beta-GP caused a prolonged elevation of ALPase, but did not affect the formation of bone nodules. Unlike Pro I-released population of cells, rat calvarial cells did not form mineralized nodules without beta-GP, and showed that a Dex-responsive period on bone nodule formation in rat calvarial cells was at preconfluency (days 0 and 1). Thus, it appeared that the Dex-induced differentiation of early OP cells in Pro IRPCs occurred during the limited period from day 9 to day 12. Pro IRPC was found to have an unique characteristic that bone nodule formation was not affected by beta-GP.

Alkaline Phosphatase↗

PubMed1989

The binding of beta-glycerophosphate to glycogen phosphorylase b in the crystal.

The binding of beta-glycerophosphate (glycerol-2-P) to glycogen phosphorylase b in the crystal has been studied by X-ray diffraction at 3 A resolution. Glycerol-2-P binds to the allosteric effector site in a position close to that of AMP, glucose-6-P, UDP-Glc, and phosphate. In this position, glycerol-2-P is stabilized through interactions of its phosphate moiety with the guanidinium groups of Arg 309 and Arg 310 which undergo conformational changes, and the hydroxyl group of Tyr 75, while the same residues and solvent are involved in van der Waals interactions with the remaining part of the molecule. Kinetic experiments indicate that glycerol-2-P partially competes with both the activator (AMP) and the inhibitor (glucose 6-phosphate) of phosphorylase b. A comparison of the positions of glycerol-2-P, AMP, glucose 6-phosphate, UDP-Glc, and Pi at the allosteric site is presented.

Adenosine Monophosphate↗

PubMed1989

Mineralization induced by beta-glycerophosphate in cultures leads to a marked increase in collagenase synthesis by mouse osteogenic MC3T3-E1 cells under subsequent stimulation with heparin.

The clonally derived mouse osteoblast-like cell line MC3T3-E1 was shown to produce latent collagenase (approximately 0.2 units/ml) under stimulation with either heparin or parathyroid hormone in confluent cultures. However, it was found that MC3T3 E1 cultures which were first induced to undergo mineralization by the addition of beta-glycerophosphate and were subsequently stimulated with heparin showed an approximately ten-fold increase in collagenase synthesis. MC3T3-E1 cell collagenase from a small sample of serum-free culture medium was purified 49-fold to a specific activity of 200 units/mg protein with a yield of 14% by heparin-sepharose affinity chromatography and ion-exchange high performance liquid chromatography. This new mineralization-primed cell culture system may be a valuable model for the study of osteoblast collagenase.

Alkaline Phosphatase↗

PubMed1984

Glycerophosphate acylation by microsomes and mitochondria of normal and dystrophic human muscle.

The incorporation of [14C]glycerophosphate into phosphatidic acid, diacylglycerol, triacylglycerol and phosphatidylcholine by microsomes and mitochondria prepared from normal and dystrophic human muscle incubated in vitro in the presence of 0.3 mmol/l CDP-choline was measured. In mitochondria only phosphatidic acid and diacylglycerol are labelled; the rate of incorporation into these two compounds showed no difference between dystrophic and normal mitochondria. In dystrophic microsomes the incorporation into phosphatidic acid was delayed and decreased. No incorporation of glycerol into diacylglycerol, phosphatidylcholine and triacylglycerol could be measured. Thus in dystrophic muscle microsomes only PA was labelled during an incubation of up to 45 min. In both types of microsomes the concentration of endogenous free fatty acids and diacylglycerol was nearly identical. The level of phosphatidylcholine was 186 and 79 nmol/mg microsomal protein in normal and dystrophic muscle microsomes, respectively. Whether the results could be explained as secondary changes was discussed. Despite some unsolved problems the conclusion was drawn that a better explanation of the results is to assume a primary defect involving microsomal-bound phosphatidic acid phosphohydrolase and possibly glycerol-P-acyltransferases.

Acylation↗

PubMed1994

A continuous spectrophotometric assay for alkaline phosphatase with glycerophosphate as substrate.

We describe a continuous coupled spectrophotometric assay for alkaline phosphatase which uses alpha- or beta-glycerophosphate as substrate, and glycerol dehydrogenase as ancillary enzyme. The glycerol liberated by alkaline phosphatase is determined by measuring the increase in absorbance at 340 nm caused by NADH formation that is combined with glycerol oxidation by the ancillary enzyme. The assay procedure was optimized using a bovine bone extract as alkaline phosphatase source.

Alkaline Phosphatase↗

PubMed1992

Inorganic phosphate added exogenously or released from beta-glycerophosphate initiates mineralization of osteoid nodules in vitro.

Rat calvaria (RC) cells grown in medium containing ascorbic acid form nodules of osteoid and cells. When 10 mM beta-Glycerophosphate (beta-GP) is added, the osteoid mineralizes in two phases: an initiation phase that is dependent upon alkaline phosphatase activity and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require added beta-GP (Bellows et al., Bone Miner 1991;14:27-40). The present experiments were performed to determine whether beta-GP is converted to inorganic phosphate (Pi) during the initiation phase of the mineralization process and whether increased Pi can replace beta-GP in the initiation phase. Measurements of Pi concentrations in the culture medium showed that during the first 8 h of the initiation phase of mineralization, 10 mM beta-GP was rapidly degraded resulting in Pi concentrations of 9-10 mM. The production rate of Pi from beta-GP was linear (r = 0.996) and the alkaline phosphatase activity in the same cultures indicated a potential for conversion of beta-GP to Pi that was greater than the actual conversion rate. The addition of 2-5 mM Pi in the absence of beta-GP also initiated mineralization. Mineralization initiated by either beta-GP or Pi progressed in the absence of added beta-GP or Pi. 100 microM Levamisole inhibited the initiation of beta-GP-induced mineralization and the conversion of beta-GP to Pi, but did not affect Pi-induced initiation of mineralization. The addition of 1-5 mM Pi to cultures in which mineralization had been initiated by 10 mM beta-GP had no significant effect on the progression phase of mineralization. Neither beta-BP nor Pi initiated 45Ca uptake in cultures without nodules (RC population I) and the histological appearance of the mineralized tissue in either phosphate source appeared identical. The present experiments show that beta-GP is rapidly and virtually completely degraded to Pi during the initiation phase of mineralization and that the addition of increased concentrations of Pi can replace beta-GP in the initiation phase of mineralization in the absence of non-specific 45Ca uptake or apparent cellular toxicity.

Alkaline Phosphatase↗

PubMed1992

Osteogenic phase-specific co-regulation of collagen synthesis and mineralization by beta-glycerophosphate in chick periosteal cultures.

Mineralized bone formation in vitro can be induced by the alkaline phosphatase substrate beta-glycerophosphate (GP). GP may not only be essential for mineralization in vitro, but could also modulate other metabolic activities of bone cells, particularly if GP is presented to these cells during different phases of development. To assess GP modulation of bone cell metabolism, biochemical and autoradiographic analyses of chick periosteal cultures treated with GP were performed. About 50% less (p less than 0.05) Type I collagen was produced in periosteal cultures treated with GP. If the fibrous portion of the periostem was microdissected from the osteogenic layer prior to culture, GP inhibition of Type I collagen synthesis was even more marked (60%: p less than 0.05). To define organic phosphate-sensitive phases of osteogenesis, cultures were exposed to GP for various time periods. Mineralization occurred reproducibly when periosteal cultures were treated with GP from the outset of the incubation period (positive control). However, if GP was added after the third day of incubation, phosphate content was the same as in positive control cultures, whereas calcium content was significantly (20%: p less than 0.05) lower. Moreover, if GP was added on day 6, there was virtually no calcium accumulation by day 12, while massive amounts of phosphate had accumulated. Taken together, these findings indicate that organic phosphates may modulate phenotypic expression of osteogenic cells, and that osteogenic cells traverse an organic phosphate-sensitive phase, after which they may be incapable of normal mineralization.

Animals↗

PubMed2005

Inhibition of glycerophosphate-dependent H2O2 generation in brown fat mitochondria by idebenone.

The established protective effect of coenzyme Q (CoQ) analogs is dependent on the location of reactive oxygen species (ROS) generation. One of these analogs--idebenone (hydroxydecyl-ubiquinone) is used as an antioxidative therapeutic drug. We tested its scavenging effect on the glycerophosphate (GP)-dependent ROS production as this enzyme was shown as a new site in the mitochondrial respiratory chain where ROS can be generated. We observed that idebenone inhibits both GP- and succinate-dependent ROS production. Idebenone and CoQ1 were found to be more efficient in the scavenging activity (IC50: 0.052 and 0.075 microM, respectively) than CoQ3 (IC50: 45.8 microM). Idebenone also inhibited ferricyanide (FeCN)-activated, GP-dependent ROS production. Our data thus extend previous findings on the scavenging effect of idebenone and show that it can also eliminate GP-dependent ROS generation.

Adipose Tissue, Brown↗

PubMed1984

Ultrastructure of bony-fish heart tissue treated with beta-glycerophosphate/lead citrate or adenosine triphosphate/lead nitrate.

The ultrastructure of bony-fish heart tissue is described when subjected to beta-glycerophosphate/lead citrate (9.2 less than or equal to pH less than or equal to 9.4) or adenosine triphosphate/lead nitrate (pH = 7.3). When treated in the former way, electron dense precipitates occur in the endocardial and myocardial endoplasmic/sarcoplasmic reticulum, Golgi apparatus, and nuclear intermembranous space. Such precipitates are absent in the control tissue and reflect therefore probably alkaline phosphatase activity. In tissue treated with adenosine triphosphate/lead nitrate the precipitates are irregularly distributed, and may also occur in the control tissue. In the present material, they therefore seem not to reflect adenosine triphosphatase activity. The results are compared and discussed with those revealed in the present study for the bony-fish intestine and with those previously reported for various mammalian tissues.

Adenosine Triphosphatases↗

PubMed1995

Application of Ca-beta-glycerophosphate for artificial apical barrier formation.

This case demonstrates an artificial apical barrier formation by using calcium-beta-glycerophosphate paste in a nonvital permanent tooth with an open apex communicating with the maxillary sinus. The long-term satisfactory outcome of this case depended on the resolution of periapical pathosis and root canal obturation without overextension.

Adult↗

PubMed2000

Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation.

This work analyses the proliferation/differentiation behaviour of human bone marrow cells cultured in alpha-minimum essential medium supplemented with 10% foetal bovine serum (standard medium) and in the presence of ascorbic acid (AA, 50 microg ml(-1)), beta-glycerophosphate (betaGP, 10 mmol) and dexamethasone (Dex, 10 nmol) under selected experimental conditions. Cultures were compared concerning cell morphology, cell growth, ALP activity and ability to form calcium phosphate deposits. Cells growing in the various experimental conditions proliferated gradually with the incubation time and presented high ALP activity. Cultures grown in standard medium and in the presence of either AA or Dex failed to form calcium phosphate deposits. Cultures grown in the presence of betaGP, betaGP + AA and betaGP + AA + Dex, i.e. in the presence of a source of phosphate ions, showed the formation of a mineralised extracellular matrix. The presence of Dex resulted in a significant induction in the ALP activity and ability to form mineral deposits. The behaviour of the various cell cultures is in agreement with previous studies stating a reciprocal and functionally coupled relationship between proliferation and differentiation, i.e. cultures grown in a medium containing betaGP presented a less proliferative but more differentiated osteoblastic cell population, as compared to cultures lacking the mineralisation process.

Alkaline Phosphatase↗

PubMedDate not supplied

A fortifier comprising protein, vitamins, and calcium-glycerophosphate for preterm human milk.

BACKGROUND: The infant's own mother's milk, fortified with proteins, minerals, and vitamins, is considered the best food for low-birth-weight infants. This paper describes the process to obtain a liquid human milk fortifier. METHODS: The fortifier comprises a protein concentrate, calcium, phosphate, and zinc salts, as well as vitamins A and D. A powdered whey protein extracted from bovine milk was concentrated from 31.5-76.8 g/100 g using repetitive dialysis. The protein concentrate was dissolved in a 0.2 M phosphate buffer pH 7.4 and mixed with calcium-glycerophosphate and calcium-gluconate, vitamins A and D, folic acid, and zinc. Each 10 mL of this liquid fortifier has 0.78 g protein, 53 mg calcium, 36 mg phosphate, and 0.93 mg zinc. RESULTS: Repetitive dialysis did not modify the protein structure as demonstrated by electrophoresis. A total of 95% of lactose content was discarded. Enriching human milk using this human milk fortifier increased the concentration per deciliter of all added nutrients; proteins increased from 1.68-2.35 g, calcium from 26-90 mg, and phosphorus, from 15-51 mg. CONCLUSIONS: A liquid human milk fortifier was successfully manufactured using a noncomplex procedure. An intake of 180-200 mL/kg/day of the fortified human milk by the premature infant would satisfy the infant's nutritional requirements and achieve expected growth and development.

Calcium Gluconate↗

PubMed1997

Post-translational modifications of meningococcal pili. Identification of common substituents: glycans and alpha-glycerophosphate--a review.

Neisseria meningitidis elaborate filamentous adhesins, pili or fimbriae, which belong to the type-4 structural group of pili also found on other bacterial pathogens such as Neisseria gonorrhoeae, Pseudomonas aeruginosa, Moraxella bovis and Dichelobacter nodosus. Meningococcal pili readily undergo structural variations which arise as a result of inter- and intra-genomic recombinational events. Structural variations often result in variations in bacterial adhesion mediated by pili. Studies on structure/function relationship of meningococcal pili have shown that pili are post-translationally modified and contain unusual covalently-linked substituents. The presence of glycans was shown by the use of specific carbohydrate labelling, chemical deglycosylation and by introducing galE mutations; the latter studies confirmed the presence of galactose on pili. Mass spectrometric and other analysis of pilin-derived tryptic peptides and linked substituents were used to determine the structure of an unusual O-linked trisaccharide, Gal beta1-4 Gal alpha1-3[2,4-diacetamido-2,4,6-trideoxyhexose]. Similar studies have also identified, perhaps a unique substituent, alpha-glycerophosphate, which is attached to Ser93 by a phosphodiester linkage.

Amino Acid Sequence↗

PubMed1998

Matrix mineralization in MC3T3-E1 cell cultures initiated by beta-glycerophosphate pulse.

MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of beta-glycerophosphate (beta-GP). In the present work, we study the influence of concentration and duration of beta-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following beta-GP pulse. With continuous treatment at 5 mmol beta-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower beta-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with beta-GP. These data indicate that, in viable cells, high doses of beta-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.

3T3 Cells↗