Dietary myo-inositol hexaphosphate prevents dystrophic calcifications in soft tissues: a pilot study in Wistar rats
Introduction
Ectopic calcification is a common disorder associated with soft tissues such as the skin, kidney, tendons and cardiovascular tissues. In arteries, calcifications are correlated with atherosclerotic plaques which increase the risk of myocardial infarction Virmani et al., 2002, Wong et al., 1995. Degenerative calcific aortic stenosis is currently the most common valvular lesion found in clinical cardiology, and it is estimated that 1–2% of the elderly population suffer from this pathology (O'Keefe et al., 1991). All mammalian extracellular fluids are supersaturated with calcium phosphate (hydroxyapatite) and consequently are metastable with respect to this solid. These crystals do not precipitate spontaneously, and under physiological conditions, crystallization takes place only in controlled situations, such as in the formation of bone and teeth. Nevertheless, uncontrolled pathological crystallization may also occur. In fact, indiscriminate crystallization in human fluids does not occur because of regulated thermodynamic (supersaturation) and kinetic factors. Thus, there are three main aspects involved in biological crystallization: supersaturation (thermodynamic factor), the presence of heterogeneous nuclei (crystallization inducers, kinetic factor), and/or crystallization inhibitors (kinetic factor). It is clear that the presence of injured tissue provides heterogeneous nucleants that serve as substrates for initial crystal formation (Valente et al., 1985). On the other hand, the action of the so-called crystallization inhibitors can obstruct or prevent crystal formation, although these processes are poorly understood. Because of their chemical structures, these substances interact with the nucleus or the crystal, provoking important disturbances in their formation and/or development and preventing the crystallization processes. The existence of crystallization inhibitors was first observed in the 1960s. Bliznakov (1965) demonstrated that some molecules reduced the rate of crystal growth of a given substance due to their adsorption on growing surfaces, and when these inhibitory mechanisms were overcome, calcium crystals precipitated and proliferated.
Myo-inositol hexaphosphate (InsP6 or phytate) is an abundant component of plant seeds. In whole grain cereals such as corn, wheat, and rice, it ranges from 1.5% to 6.4%, while defatted and dehulled oilseed meals such as soy, peanut, and sesame meals contain 1.5% or more of the compound. In most seed types the InsP6 is associated with calcium and magnesium ions (the so-called phytin) and is not equally distributed. For example, the endosperm of wheat and rice kernels are almost devoid of InsP6 as it is concentrated in the germ and aleuronic layers of cells of the kernel and in the bran or hull. Corn differs from most cereals as almost 90% of InsP6 is concentrated in the germ portion of the kernel as in the carob germ. The levels found in blood and mammalian tissues clearly depended on their dietary intake Grases et al., 2001a, Grases et al., 2001b. Because we demonstrated that this molecule exhibited a potent capacity as crystallization inhibitor of calcium salts in urine Grases et al., 1996, Grases et al., 1998a, Grases et al., 1998b, we proposed in the present paper to study the effects of dietary InsP6 on artificially provoked dystrophic calcifications in soft tissues. Etidronate, a bisphosphonate class of drug Fleisch, 1997a, Fleisch, 1997b, was included for comparative purposes because of its crystallization-inhibitory effects in both hydroxyapatite and brushite formation Fleisch, 1997b, Grases et al., 2000c.
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Animals, diets and experimental design
Twenty-four male Wistar rats of about 300 g from Harlan Iberica s.l. (Barcelona Spain) were acclimatized for 10 days. They were fed normal rat chow ad libitum and had free access to drinking water. Housing was in groups of three rats in Plexiglas cages. Environmental conditions were temperature 21 ± 1 °C, humidity 60 ± 5%, and light-dark periods 12:12 hours. They were randomly assigned into four groups of six rats each. The diets used were 4068.02 Reference Diet (HopeFarms BV, Woerden, The
Statistics
Values in the tables and figures are expressed as means ± (SE). One-way ANOVA was used to calculate significance of differences between groups. Student t-test was used to assess differences of means. The SPSS for the Windows program was used for statistical computations. Values for p are given in Fig. 2 and Table 2, Table 3. A value of p < 0.05 was considered to assess statistical significance.
Results
Blood levels of InsP6 in the four groups of rats are shown in Table 2. Animals consuming during 31 days the purified diet to which a 1% of InsP6 (as sodium salt) was added showed InsP6 blood levels of 0.393 ± 0.013 μM which were similar to those found in rats consuming a standard UAR-A03 diet (Grases et al., 2001e). In animals consuming only the purified diet lacking InsP6, the InsP6 blood levels decreased (0.026 ± 0.006 μM). Animals consuming the same purified diet to which etidronate was
Discussion
Dystrophic mineralization commonly occurs in soft tissues as a consequence of injury, disease, and aging with or without degenerative changes evident in the tissue. Although most soft tissues can undergo calcification, skin, kidney, tendons and cardiovascular tissues appear predisposed to develop this pathology (Anderson and Morris, 1993). Dystrophic calcifications, also termed ectopic calcifications, are typically composed of calcium phosphate salts, such as hydroxyapatite, but can also
Acknowledgements
This work was supported by the Conselleria d'Innovació i Energia del Govern de les Illes Balears and by the project BQU 2003-01659 of the Spanish Ministry of Science and Technology. One of the authors, J.P., expresses his appreciation to the Spanish Ministry of Education, Culture and Sport for a fellowship of the FPU program.
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