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. 2004:6:263-267.
doi: 10.1251/bpo97. Epub 2004 Dec 16.

A simple and non-radioactive technique to study the effect of monophosphoesters on matrix vesicle-mediated calcification

Rama Garimella  1 , Joseph B Sipe, H Clarke Anderson
Affiliations

A simple and non-radioactive technique to study the effect of monophosphoesters on matrix vesicle-mediated calcification

Rama Garimella et al. Biol Proced Online. 2004.

Abstract

A simple and non-radioactive technique based on O-cresolpthalein complexone assay was developed to study in vitro non-radioactive calcium ((40)Ca) deposition by isolated matrix vesicles. Using this technique, the effect of various phosphoester substrates including ATP, AMP and beta-GP on in vitro MV-calcification was studied. O-cresolpthalein complexone assay with non-radioactive calcium demonstrated that AMP or beta-GP were more effective in promoting calcium deposition by isolated MVs than ATP. The application of this non-radioactive technique, which is highly sensitive and simple, would offer a useful alternative approach to the routinely used radiometric biomineralization assay which employs radioactive (45)Ca.

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Figures

Fig. 1
Fig. 1. Electron micrograph of mineralizing matrix vesicles showing radial clusters of hydroxyapatite like mineral (X 110,000).
The dark arrows show needle shaped hydroxyapatite mineral while the grey arrow shows intact MV. MVs isolated from growth plates of rachitic rats were calcified upon exposure to calcifying buffer containing physiological concentrations of Ca2+ and Pi. The calcified MVs were fixed in 2.5% glutaraldehyde, post fixed in 1% osmium tetraoxide, dehydrated, embedded in Spurr's low viscosity epoxy resin. Thin sections were cut and stained with uranyl acetate and lead citrate, and photographed using a Zeiss EM IOA electron microscope.
Fig. 2
Fig. 2. Flow diagram showing the isolation of rat growth plate matrix vesicles (as described in (3) and (21)).
Fig. 3
Fig. 3. Standard curve for o-cresolopthalein complexone assay.
Fig. 4
Fig. 4. Comparison of in vitro calcification profile of isolated MVs in the presence of 1mM ATP, 3mM AMP and 3 mM β-GP using O-CPC assay.
The calcium concentration is first obtained for each of the samples using calcium standard. The relative values are used to compare changes in the calcium deposition ability relative to the control reference sample (MVs in the absence of phosphoester substrates). Values are expressed as means ± S.D. from five different MV preparations. Double asterisks are assigned to indicate the statistical significance (P<0.05) with respect to control. Single asterisks are assigned to indicate the statistical significance (P<0.05) between groups (MV+ATP vs. MV+AMP, and MV+ATP vs. MV+ β-GP).
Fig. 5
Fig. 5. Schematic diagram showing the metabolism of ATP and AMP and the effect of products of their hydrolysis on mineralization.
ATP is hydrolyzed by nucleoside triphosphate pyrophosphohydrolase (NTPPPH) to AMP and inorganic pyrophosphate (PPi), which get further degraded to yield inorganic phosphate (Pi) by alkaline phosphatase (ALP). The inorganic phosphate thus generated forms apatite in the presence of calcium of the extracellular fluid. Accumulation of excess PPi results, in the inhibition of apatite deposition.
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References

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