Rafael M. Prieto

Variation of InsP4,InsP5 and InsP6 levels in tissues and biological fluids depending on dietary phytate

Due to the increasing interest of InsP(6) on human health, the aim of this paper is to compare the levels of highly phosphorilated inositols (InsP(4), InsP(5) and InsP(6)) in organs and biological fluids of rats and to study the influence of the presence and absence of InsP(6) in diets. Thus, for this purpose, the variation of InsP(4), InsP(5) and InsP(6) levels in organs and biological fluids of rats submitted to two different diets were studied. In the AIN-76A diet no InsP(6) was present, yet the other was a 1% InsP(6) modified diet (AIN-76A + 1% InsP(6)). The highest InsP(4), InsP(5) and InsP(6) levels were found to be 10-fold superior in the brain than those found in the kidney. When the InsP(6) was eliminated from the diet, the InsP(6) levels decreased dramatically (97.2% in kidney, 89.8% in brain, 100% in bone, 90.5% in plasma and 98.1% in urine), the InsP(5) levels showed an important decrease (61.2% in kidney, 45.5% in brain, 28.1% in bone, 30% in plasma and 88.6% in urine) and the InsP(4) levels in organs only changed slightly. From these results, it can be deduced that the majority of InsP(6) present in the organism is of dietary origin and its endogenous synthesis is not important. According to the results, it can be evidenced that the endogenous synthesis of InsP(5) can occur, besides InsP(6) can be transformed by enzymatic dephosphorilation in InsP(5).

Effects of exogenous inositol hexakisphosphate (InsP6) on the levels of InsP6 and of inositol trisphosphate (InsP3) in malignant cells, tissues and biological fluids

InsP(6) is abundant in cereals and legumes. InsP(6) and lower inositol phosphates, in particular InsP(3), participate in important intracellular processes. In addition, InsP(6) possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabeled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP(6) and InsP(3) in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabeled total InsP(3) and InsP(6) (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP(6). When rats were fed on a purified diet in which InsP(6) was undetectable (AIN-76A) the levels of InsP(6) in brain were 3.35 +/- 0.57 (SE) micromol.kg(-1) and in plasma 0.023 +/- 0.008 (SE) micromol.l(-1). The presence of InsP(6) in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP(6)-sufficient diet (AIN-76A + 1% InsP(6)), the levels of InsP(6) were about 100-fold higher in brain tissues (36.8 +/- 1.8 (SE)) than in plasma (0.29 +/- 0.02 (SE)); InsP(6) concentrations were 8.5-fold higher than total InsP(3) concentrations in either plasma (0.033 +/- 0.012 (SE)) and brain (4.21 +/- 0.55 (SE)). When animals were given an InsP(6)-poor diet (AIN-76A only), there was a 90% decrease in InsP(6) content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP(3). In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP(6) contents were 16.2 +/- 9.1 (SE) micromol.kg(-1) for MDA-MB 231 cells and 15.6 +/- 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP(3) (4.8 +/- 0.5 micromol.kg(-1) and 6.9 +/- 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP(6) resulted in a 2-fold increase in the intracellular concentrations of total InsP(3) (9.5 +/- 1.3 (SE) and 10.8 +/- 1.0 (SE) micromol.kg(-1) for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP(6) levels. These results indicate that exogenous InsP(6) directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP(3) levels. Although a similar fluctuation of InsP(6) concentration was not seen in human malignant cell lines following InsP(6) treatment, an increased intracellular levels of total InsP(3) was clearly observed.

Dietary phytate and mineral bioavailability

The relation between the dietary phytate (InsP6), mineral status and InsP6 levels in the organism, using three controlled diets (AIN-76A, AIN-76A + 1% phytate, AIN-76A + 6% carob seed germ), are studied. AIN-76A is a purified diet in which InsP6 is practically absent. No important or significant differences in the mineral status (Zn, Cu, Fe) of blood, kidneys, liver, brain and bone, were observed, except iron in the brain. Thus, the amounts of iron found in the brain of rats fed AIN-76A + 1% InsP6 were significantly inferior to those found in rats fed AIN-76A diet. The amounts of InsP6 found in organs of rats fed AIN-76A diet became very low or even undetectable while the ones found in rats fed diets that contained 1% and 0.12% (AIN-76A + 6% carob seed germ) InsP6, were considerably higher and similar. Moreover the majority of rats fed AIN-76A diet exhibited calcifications at the corticomedullary junctions, whereas no calcifications were detected in rats fed the other two diets. From these results, it can be deduced that there was no important adverse effects on mineral status as a consequence of the presence of InsP6 in the studied diets. Besides, considering that a 0.12% InsP6 contained in the AIN-76A purified diet through the addition of a 6% of carob seed germ to this diet, produced the same beneficial effects as the direct addition of a 1% of InsP6 and no negative effects on mineral status was observed, it can be concluded that the value of the presence of InsP6 at adequate amounts in the diet is remarkable and must be favourably considered.

Urinary pH and renal lithiasis

Formation of calcium oxalate crystals, either as monohydrate or dihydrate, is apparently unrelated to urinary pH because the solubilities of these salts are practically unaltered at physiologic urinary pH values. However, a urinary pH <5.5 or >6.0 may induce uric acid or calcium phosphate crystals formation, respectively, which under appropriate conditions may induce the development of the calcium oxalate calculi. We assessed the relationship between the urinary pH and the formation of different types of calculi. A retrospective study in 1,478 patients was done. We determined the composition, macrostructure, and microstructure of the calculi and the urinary pH, 50.9% of calcium oxalate monohydrate unattached calculi were present in patients with urinary pH <5.5. We found that 34.1 and 41.5% of calcium oxalate dihydrate calculi were present in patients with urinary pH <5.5 and >6.0, respectively. Infectious calculi were found primarily in patients with urinary pH >6.0 (50.7%). Only calcium oxalate monohydrate papillary calculi were associated with urinary pH between 5.5 and 6.0 (43.1%). Urine of pH <5.5 shows an increased capacity to develop uric acid crystals, which can act as a heterogeneous nuclei of calcium oxalate crystals. In contrast, urine of pH >6.0 has an increased capacity to develop calcium phosphate crystals, which can act as a heterogeneous nuclei of calcium oxalate crystals. Oxalate monohydrate papillary calculi were associated to pH between 5.5 and 6.0 because the injured papilla acts as a heterogeneous nucleant. Consequently, measurement of urinary pH may be used to evaluate the lithogen risk of given urine.

Phytate inhibits bovine pericardium calcification in vitro

OBJECTIVE The present study examined the inhibitory effects of pyrophosphate, etidronate, and phytate on bovine pericardium calcification in vitro. METHODS Bovine pericardium was glutaraldehyde fixed and then placed in a flow chamber in the presence of a synthetic physiological fluid alone (control) or the fluid plus various concentrations of pyrophosphate, etidronate, or phytate. Following a 96-h incubation, fragments were removed and assayed for calcification by measuring calcium and phosphorus levels. RESULTS The data indicated that both pyrophosphate and etidronate at 1 mg/l (5.75 and 4.95 microM, respectively) inhibited bovine pericardium calcification, whereas neither agent had an effect at 0.5 mg/l (2.87 and 2.47 microM, respectively). Phytate was the most potent inhibitor of calcification, and the effects of this agent were apparent at levels as low as 0.25 mg/l (0.39 microM). CONCLUSIONS While pyrophosphate, etidronate, and phytate were all able to inhibit bovine pericardium calcification in vitro, phytate was found to be the most effective.

Phytotherapy in a rat model of hyperoxaluria: the antioxidant effects of quercetin involve serum paraoxonase 1 activation

Serum paraoxonase 1 (PON1) has been reported to be an important contributor to the antioxidant and anti-inflammatory activities of HDL, avoiding LDL oxidation. The activity of this enzyme is reduced in patients with renal insufficiency, caused by elevated oxidative stress and disturbances of apolipoprotein metabolism. Therapeutic utilization of antioxidants to control renal oxidative stress may be an effective therapy in renal protection. The aim was to investigate the protective effects of several antioxidant compounds against the oxidative stress associated to renal failure induced by ethylene glycol (EG), focusing on the possible role of serum PON1 activity. Fifty-four male Wistar rats were randomly assigned to six groups (n = 9): an untreated control (C) group, an EG-treated group, a catechin (CAT)-treated group, an epicatechin (EPI)-treated group, a quercetin (QUE)-treated group and a folk herbal extract (FHE)-treated group. After 16 d of treatment, calcium oxalate lithiasis was induced in the rats using EG. After eight days (treatment + EG), the animals were sacrificed. EG treatment impaired kidney composition, increased oxidative damage, and decreased serum paraoxonase and arylesterase activities. CAT, QUE and the FHE Fagolitos improved oxidative status by enhancing antioxidant defenses – superoxide dismutase and PON1 activities – and reducing oxidative damage, thus reinforcing the idea of a possible role of PON1 in the protective effects of QUE against the deleterious consequences of oxidative stress in kidney.

Effect of Tetracalcium Dimagnesium Phytate on Bone Characteristics in Ovariectomized Rats

The aim was to evaluate the influence of dietary Ca-Mg-phytate consumption on the bone characteristics of ovariectomized rats, an animal model for postmenopausal osteoporosis. Twenty ovariectomized female Wistar rats were randomly assigned to two groups fed, respectively, with a non-phytate diet (AIN-76A) or the same diet enriched with 1% phytate (as the calcium magnesium salt, phytin). After 12 weeks of feeding the rats were sacrificed, and both femoral bones and L4 vertebra were removed from each rat. Bone mass, length, width, volume, and mineral density were measured, and the phosphorus, calcium, magnesium, and zinc contents of bones were determined. Deoxypyridinoline (a bone resorption marker) was measured in urine, and osteocalcin (a bone formation marker) was measured in serum. The calcium and phosphorus contents and bone mineral density were significantly higher in both femoral bones and L4 vertebra for phytate-treated rats in comparison to rats in the non-phytate group. Deoxypyridinoline was significantly increased in rats in the non-phytate treatment group. Ca-Mg-phytate consumption reduces bone mineral density loss due to estrogen deficiency. Thus, phytate exhibits effects similar to those of bisphosphonates on bone resorption and may be of use in the primary prevention of osteoporosis if larger studies in humans confirm these findings.

A potential role for crystallization inhibitors in treatment of Alzheimer’s disease

Melatonin is a hormone synthesized from the neurotransmitter serotonin and is found mainly in the pineal gland. Melatonin has been suggested to have several properties, acting both as an antioxidant and a neuroprotective agent. Melatonin synthesis decreases with age in all humans, but this decline is more pronounced in Alzheimers patients. In fact, melatonin inhibits the formation of beta-amyloid protein. The mechanism responsible for this decline has not been fully elucidated, although it is known that the human pineal gland calcifies with age. Such calcification necessarily implies the existence of a tissue injury that, if not reabsorbed by the immune system, will act as heterogeneous nucleant for hydroxyapatite and will induce calcification. For this reason, it is hypothesized that a lack of inhibitors of calcium salt crystallization, such as pyrophosphate and phytate, will favor calcification. Therefore, the absence of crystallization inhibitors may be a risk factor for development of Alzheimers disease, and this hypothesis should be evaluated.

Non-infectious phosphate renal calculi: Fine structure, chemical and phase composition

Abstract Background. Chemical composition of internally non-homogeneous phosphate stones should be related to the conditions prevailing during the formation of each individual part. Objective. The object of this paper was to provide a detailed study of phosphate stone composition on the micro- and macro-scales. Methods. Fine inner structure, chemical and phase composition of 10 phosphate calculi from different patients were determined by chemical (wet) analysis, observation by scanning microscope, semi-quantitative determination of Ca, Mg, P and C by energy dispersive X-ray and by X-ray diffraction. Results. Eight calculi are formed by amorphous calcium phosphate and two by hydroxyapatite. Magnesium was inversely related to Ca/P ratio. Point chemical composition of solid phase varies in wide limits, i.e. composition of calculus interior is highly inhomogeneous on the microscale. All studied calculi contained an abundance of organic matter incorporated in their volume; the content of carbon was double the calcium content in molar quantities. Conclusions. Phosphate renal calculi with the low Ca/P molar ratio predominantly consist of amorphous calcium phosphate whereas those with a high Ca/P molar ratio are composed of poorly crystalline hydroxyapatite which can be partially carbonated. Magnesium may be an inhibitor of HAP formation from urine. Abundant organic matter incorporated into the calculus volume indicates its decisive role at stone formation. Variable point composition of stones implies widely varying conditions during their development.

A new device for simple and accurate urinary pH testing by the Stone-former patient

PurposeUrinary pH is an important factor linked to renal stone disease and a useful marker in the treatment of urolithiasis. Although the gold standard for measuring urinary pH utilizes a glass electrode and a pH meter, at present dipstick testing is largely used to estimate urinary pH. However, the accuracy and precision of this method may be insufficient for making clinical decisions in patients with lithiasis. The aim of this study is to describe a new device for urinary pH testing.MethodsThe device includes a pH sensor based on differential measurement of an ISFET-REFET pair. The drawbacks associated with this type of configuration, namely short lifetime and manual fabrication, have been overcome in the prototype. An automatic one point calibration is performed when turning on the system. Two buffer solutions were utilized to determine the intra- and inter-day precision of the device. The pH of 30 fresh human urine samples was measured using a pH-meter, a dipstick and the new electronic device.ResultsIn some cases, dipstick measurements differed from those of the pH meter by more than 0.40 units, a clinically relevant discrepancy, whereas none of the measurements made with the new electronic device differed from the results of the pH-meter by more than 0.1 pH units.ConclusionsThis new electronic device has the possibility to be used by stone-formers to control their urinary pH at home, increasing the tools available for stone prevention and prophylaxis.