Ca Deposition Research Articles

Evidence for an intestinal mechanism in hypercalciuria of spontaneously hypertensive rats.

To define the mechanism for the hypercalciuria in spontaneously hypertensive rats (SHR), Ca clearance was evaluated in fasted 23-wk-old SHR and normotensive Wistar Kyoto (WKy) controls. There was no exaggerated calciuria before or after parathyroidectomy. Ca balance was therefore measured in the nonfasted animals, which revealed hyperabsorption in SHR of both sexes with increments 10-fold that of Ca excretion, supporting the primacy of intestinal hyperabsorption. In situ duodenal Ca uptake was also increased in the SHR. Parathyroidectomy did not affect the hyperabsorption. Hypercalcemia (total and ionized) and hypercalciuria in SHR associated with reduced adenosine 3',5'-cyclic monophosphate excretion, were abolished by fasting. Correction of hypertension for 6 mo failed to abolish the hypercalciuria. Bone Ca deposits were increased in 1-yr-old SHR. Ten-week-old SHR, in contrast, displayed mild malabsorption. Our data do not support the "renal leak" hypothesis. Instead, the adult SHR is characterized by increased Ca retention due to primary hyperabsorption, absorptive hypercalciuria, and increased bone Ca deposition. These phenomena are independent of sex, parathyroid hormone, and treatment of the established hypertension.

Time Course of Changes in Various Factors Associated with the Injury of Rat Testis Induced by CdCl<SUB>2</SUB>

Long-term experiments were carried out, for up to 300 d after CdCl2 injection, to investigate the mechanism of the testis injury induced by CdCl2. The results are summarized as follows ; 1) Testis weight and lipoperoxide concentration were increased significantly at 3 d after CdCl2 injection, and thereafter decreased steadily. The values at 300 d were very much lower than those of control group. 2) Although hemoglobin content in the testes treated with CdCl2 was significantly higher at 3 d, presumably because of the intensive hemorrhage in situ, it decreased gradually and the value at 300 d after injection had returned almost to the control level. 3) In the testis at 3 d after CdCl2 injection, edematous was found in the interstitial tissue numerous neutrophils had migrated into the seminiferous tubules and the tubules were contracted. These changes, however, disappeared as time elapsed after CdCl2 injection. By 300 d, the seminiferous tubules were filled with materials which had a high concentration of Ca and P. The concentration of Ca in the interstitial tissues was also high. The enhancement of Ca deposition was observed in the testes of rats 3 d after CdCl2 injection. 4) Although the activities of serum GOT and GPT were significantly higher at 3 d after CdCl2 administration, they returned to the control level by 15 d after the injection and thereafter they showed normal values throughout the remainder of the observation periods. On the contrary, Al-P activity decreased significantly at 3 d after the injection. However, the enzyme activity returned to the control level at 90 d.

Role of calcium and free fatty acids in epinephrine-induced myocardial necrosis

A possible mechanism by which large doses of catecholamines produce myocardial necrosis was investigated. Male Sprague-Dawley rats, 275 to 325 g in weight, were injected once, sc, with 3 mg/kg epinephrine (E) or infused iv for 1 hr with E at a rate of 1.2 or 1.7 μg/min, and also injected iv with either 45Ca or [ 3H]palmitic acid ( 3H-PA) at the same time as or at various periods of time after E administration but exactly 0.5 or 1 hr before death. Controls were injected with saline solution. Heart/plasma ratios of radioactivity ( H P ) were determined. The ratios increased in the case of both 45Ca and [ 3H]PA within 0.5 hr after E, reached peak values after 18 to 24 hr with 45Ca and 3 to 6 hr with [ 3H]PA, and remained above values for the controls for at least 72 hr with 45Ca and 48 hr with [ 3H]PA. The rate of 45Ca influx into heart 20 hr after E administration paralleled the severity of the myocardial damage that had been produced. When 45Ca and E were injected simultaneously, H P increased progressively with time to 30 times control values, indicating the accumulation and retention of Ca in the heart. Under the same conditions, H P values with [ 3H]PA also rose but remained constant at a level two to three times that in controls. Total cardiac free fatty acids (FFA) rose slightly and remained constant at the elevated level. It was not possible to distinguish a given point in time at which the increase in either Ca or FFA influx, initially due to the normal pharmacological effect of E, began to occur as a consequence of damage produced by the latter. It is concluded that high concentrations of catecholamines promote the deposition of Ca and FFA in myocardial cells in various forms, and that the deposition of these substances as soaps in the plasma membranes may cause permeability changes that lead to cell injury.

Non uniform effect of estradiol on the rat skeleton.

Pharmacological doses of estradiol administered to rats increased the Ca mass of their femurs through a significant reduction in the rate of Ca resorption. On a body basis, the same animals showed increased rates of skeletal Ca deposition and resorption and no effect on the skeletal mass. The non uniform effect of estradiol on the skeleton, if confirmed in the human being, may limit the applicability of estrogenic treatment.

Action of phosphorus on calcium release in isolated perfused rat tails

A bone perfusion system using isolated rat tails was developed to study the action of inorganic phosphorus (Pi) on calcium (Ca) release in bone. Rats were either pre-labelled with 45Ca to study calcium removal from bone or 45Ca was added to the perfusate to study Ca deposition into bone. When perfusate Pi was increased from 3.5 to 5.25 mg/dl, a marked inhibition of 45Ca release was demonstrated. 85Sr microspheres, used to demonstrate differences in the flow of perfusate between bone and soft tissue, showed that the Pi effect was not due to alterations in perfusate flow to bone. Increasing the perfusate Pi concentration from 3.5 to 7 mg/dl did not increase 45Ca deposition into bone, nor did it affect baseline or PTH-stimulated cyclic AMP production. The Pi effect was inhibited by reducing the perfusate temperature to 4 degrees C, by using an anerobic perfusate, adding iodoacetate to the perfusate, and finally, by pre-treating the rats with actinomycin D prior to the experiment. Thus, in this model an increase in the Pi concentration of the perfusate inhibits Ca release from bone but does not increase Ca uptake by bone. The inhibitory effect appears to be dependent on normal cell metabolism.

The effects of diltiazem in dystrophic hamsters.

Calcium accumulates in muscles of dystrophic hamsters (DH) and patients with Duchenne muscular dystrophy. Various Ca antagonists were beneficial to the cardiomyopathy of DH, but had only minor effects on skeletal muscle. We administered a new Ca antagonist, diltiazem, 25 mg/kg/day orally to normal and dystrophic hamsters from ages 37 to 92 days. We observed a marked reduction in muscle Ca in DH treated with diltiazem: 73% in the heart, 61% in the diaphragm, and 48% in the rectus femoris. Plasma CK was significantly lower (by 37%) in treated DH, while the elevated rate of noncollagen protein synthesis in the diaphragm was not diminished. Histologically, the most important change was a reduction in Ca deposits in the heart. Diltiazem was well-tolerated by all animals and did not modify Ca content in normal hamsters. This study suggests that diltiazem may have therapeutic value in those conditions that are accompanied by excessive accumulation of Ca in tissues, such as muscular dystrophy.

Turnover of Bone Zinc during Normal and Accelerated Bone Loss in Rats

The ability of the skeleton to serve as a reservoir of available zinc was investigated in weanling and 7-week-old rats. Bone turnover was measured by the loss of previously incorporated 3H-tetracycline (TC), in conditions of normal turnover accompanying growth or accelerated loss produced by calcium deficiency. The 4-week studies involved four experimental groups per age group (all combinations of high and low calcium, high and low zinc). By measuring loss of 3H-TC and whole bone (humerus) contents of Zn and Ca, it was possible to calculate the deposition and resorption of Zn and Ca during the experimental period. Several conclusions emerged from the study: 1) availability of Zn from the skeleton was dependent on the rate of bone resorption, but not on Zn status; 2) redeposition of Zn in the skeleton following resorption was extensive, and independent of the rate of bone mineral deposition; and 3) in Ca deficiency there was an increased deposition of Zn, suggesting a partial substitution of Zn for Ca in bone mineral. The results support the concept that the skeleton does not serve as a reservoir for Zn. Very little Zn is made available by bone resorption, and even then Zn is lost only in a passive manner that is dependent on, but does not influence, bone turnover. The availability of Zn from the skeleton is not mediated by Zn status.zinc turnover bone

Coupling of ATP pyrophosphorolysis to transport of Ca 2+ in mitochondria

In the coupling of ATP pyrophosphorolysis to Ca 2+ transport in beef heart mitochondria, for each molecule of ATP cleaved, one proton is released and one Ca 2+ is transported into the interior space. With the use of tritium labelled ATP, it could be shown that ATP is pyrophosphorylyzed into a species equivalent to Pi that moves inward, and a species equivalent to ADP that is extruded into the aqueous space on the exterior of the mitochondrion. The species equivalent to Pi has been identified as a negatively charged form of Pi (PO −) and the species equivalent to ADP as a positively charged form (ADP +). The inward flow of PO − is coupled to the inward flow of Ca 2+ in 1:1 stoichiometry—a token that Ca 2+ must enter in the form of Ca 2+A −, where A − is a monovalent anion. During ATP pyrophosphorolysis protons are released on the I side and taken up on the M side—one proton for each molecule of ATP cleaved. The alkalinization of the matrix space leads to the deposition of Ca 3(PO 4) 2 and to the extrusion of the two species released by this deposition (Pi, K +). Two thirds of the PO − is trapped as Ca 3(PO 4) 2 in the matrix space and one third is extruded into the external space. The extrusion of K + provides a mechanism by which protons can be continuously brought into the matrix space to sustain a high rate of coupled pyrophosphorolysis of ATP. The coupling pattern for Ca 2+ transport driven by ATP pyrophosphorolysis is identical with the corresponding pattern for Ca 2+ transport driven by electron transfer. This identity is suggestive that coupling mediated by the Fo-F 1 system and coupling mediated by electron transfer complexes are mechanistically indistinguishable.

Calcium and Magnesium Levels in the Annual Rings of ‘McIntosh’ Apple Wood1

Abstract Analyses were made of Ca and Mg in a consecution of annual rings of 3 mature ‘McIntosh’ apple (Malus domestica Borkh.) orchard blocks in New York (acid rain region) and also 3 mature ‘McIntosh’ blocks in British Columbia (arid-irrigated region) in an attempt to assess the long range effects of acid rain on Ca levels in apple trees. Differences in patterns of Ca and Mg deposition in the wood did not appear to be caused by acid rain.

Calcification of isolated matrix vesicles and reconstituted vesicles from fetal bovine cartilage.

Ca deposition by isolated matrix vesicles from fetal calf growth plate cartilage and by a deoxycholate extract from matrix vesicles that included their phosphatase was studied under defined in vitro conditions. Electron microscopy showed that after removal of deoxycholate and lyophilization of the vesicle extract, new vesicles were reconstituted, often with multiple membrane layers. Both intact calf vesicles and reconstituted vesicles initiated Ca deposition maximally when supplied with ATP, GTP, CTP, or UTP. Only nucleoside triphosphates supported Ca deposition well; mono- and diphosphoesters, although hydrolyzed, were ineffective as substrates. Nucleoside triphosphates supported Ca deposition even if the final [Ca] X [P] reached in the reaction mixture was below a metastable level (3.5 mM2), suggesting that matrix vesicles or reconstituted vesicles promote calcification by localizing Ca or PO4 or both. ATP or GTP supported Ca deposition readily at concentrations ranging from 0.25 to 1.0 mM but, at 2.5 and 5.0 mM, Ca deposition was inhibited. The ATPase of intact matrix vesicles and reconstituted vesicles was stimulated by addition of Ca2+ and Mg2+. Ca deposition did not require additional Mg2+. These results lend support to the hypothesis that matrix vesicles and their phosphatases play an important role in mineralization.

872 CALCIUM DYNAMICS IN INFANCY

Calcium (Ca) disorders and therapeutic effectiveness can be assessed by measurements of bone dynamics using stable tracer methods. A pulse of IV Ca-46 is followed by a 9-15 day continuous feeding of Ca-48. Ca isotopic abundances in serum, urine, and feces are determined by thermoionization mass spectrometry (0.5% precision). Kinetic parameters (mg/kg/d) are calculated by nonlinear, weighted least squares analysis and growth is compensated for by using expanding pool sizes. Two infants (Inf.) had Ca turnover rates 12 times that of a control adult (Ad); bone Ca deposition (vo+) and resorption (vo-) were determined with 2 to 5% precision. Two infants with osteogenesis imperfecta (OI) were studied before (B) and while taking ascorbic acid (C). Inf show excessive endogenous fecal (vf) to urinary (vu) Ca excretion as compared to adults, suggesting a major role for vf in regulating Ca balance in Inf. An Inf with OI had similar Ca dynamics except for a decrease in bone Ca fluxes. Ascorbic acid, as suggested for clinical use, had no effect on bone calcium balance.

Surface layer phenomena in in-vitro early caries-like lesions of human tooth enamel

Surface layer properties of artificial carious lesions produced in lactate buffers containing diphosphonate (MHDP) were studied by X-ray diffraction, optical and scanning electron microscopy, microradiography, microprobe and chemical analysis. The surface layer thickness ( d) was independent of pH and concentration of lactate HL, but dependent on the concentration of MHDP. A minimum concentration of MHDP was required for surface layer formation, and a maximum was indicated, beyond which no further increase in concentration affected it. After initial attack, MHDP largely protects the surface. The surface layer grows to an equilibrium thickness when the rate of loss equals the rate of deposition of Ca and PO 4 from subsurface dissolution into defect positions of the existing original crystals.

Rote of adenosine triphosphatase, phospholipids, and vesicular structure in the calcification of isolated and reconstituted matrix vesicles

Calcium deposition in isolated rabbit matrix vesicles can be supported by hydrolysis of a variety of phospheesters. However, with fetal calf matrix vesicles only nucleoside triphosphates can support calcification. Ca deposition occurs readily within 2 hours in the presence of physiological concentrations of Ca 2+ and HPO 5 2− and a minute quantity of isolated matrix vesicles (about 20 μg protein). Vesicles solubilized with deoxycholate (DOC) will not sediment at 250,000 x g. Such DOC-dispersed vesicles can be reconstituted into larger vesicles after DOC removal by lyophilization, and resuspension in H 2O. These reconstituted vesicles can deposit Ca in the presence of ATP and a calcifying solution. The matrix vesicle ATPase can be readily solubilized by DOC-butanol treatment and subsequent removal of the organics. The solubilized ATPase failed to deposit Ca even though the enzyme activity was made equivalent to that of matrix vesicles. Present evidence suggests that a vesicular structure with an incorporated specific phosphatase is required to produce a microenvironment suitable for concentration of calcium and phosphate to enable mineral initiation.

A new method to measure 45calcium accumulation by matrix vesicles in slices of rachitic growth plate cartilage

Slices of rachitic growth plate cartilage have proven a difficult tissue in which to study 45Ca uptake because, even in rickets, there exists an area of preformed mineral at the base of the growth plate. This extraneous mineral accumulates 45Ca through exchange and apatite crystal proliferation when exposed to a calcifying solution, thus creating a background of radioactivity which obscures 45Ca uptake into vesicle-initiated mineral. We have succeeded in removing the preformed mineral, and hence much background radioactivity by exposing slices of rachitic rat epiphyseal plate to 30 mM EGTA for 30 minutes prior to incubation in a calcifying solution. After EGTA, an alizarin-red stainable extraneous mineral line is removed, and then following incubation all newly formed mineral is deposited primarily in vesicle-containing areas. Thus, it was possible to enhance significantly the 45Ca uptake into living versus heat inactivated growth plates. Preliminary studies with the EGTA-treated rachitic cartilage also indicate that supplying a matrix vesicle phosphatase substrate such as beta-glycerophosphate (BGP) rather than supplying orthophosphate (P i) in the incubation mixture leads to more active 45 Ca deposition. We conclude that BGP can be hydrolyzed by vesicle phosphatases to supply P i for nascent mineral formation, and that our new system promises to be useful in analyzing agents which may stimulate matrix vesicle calcification.

Changes in cellular composition during magnesium deficiency.

Mg deficiency increased the water content of the liver, kidney, heart and thigh muscle in the rat and decreased the proportion of nitrogen in the dry matter of the same tissues. Changes in the concentration of metals also occurred. 2. Cellular fractional indicated that the Mg and K depleted in liver occurred primarily in the heavy-mitochondrial and microsomal fractions respectively. The calcification of liver and kidney was due to preferential deposition of Ca in the heavy-mitochondrial fraction. 3. The proportion of cellular nitrogen present in the heavy-mitochondrial and microsomal fractions was markedly decreased in the liver and kidney of the Mg-deficient rats, and the proportion in the supernatant fraction increased. 4. Electron microscopy revealed that the cross-sectional areas of liver cells and all their mitochondria were decreased in Mg deficiency. The number of mitochondrial per cell was decreased even more severely and the average area of a mitochondrion was greater in deficient rats than in control animals. 5. The significance of these observations is discussed in relation to the location of the primary metabolic disturbance during Mg deficiency.

Influence of Pre-Laying Dietary Calcium upon Tibial 45 CA Uptake in 20-Week-Old Pullets

At 5, 10 and 15 weeks of age, 20 pullets were changed from a 1.30% Ca diet to one containing 0.65 or 2.60% Ca. At 19 weeks of age, half these pullets were changed to a 2.00% Ca diet. Ten pullets each were fed the 0.65% and 2.60% Ca diets from hatching to the termination of the experiment. At 20 weeks of age, all the pullets were administered 45 Ca into the crop and sacrificed 24 hours later. Changing the diet had no significant effect on 45 Ca deposition in the tibia regardless of the time the change was made as long as all the groups compared were fed the same final diets. However, the pullets fed the 0.65% Ca diet deposited more 45 Ca in their tibias than did those fed the 2.60 or 2.00% Ca diet. Adaptation to calcium fed prior to laying was not the result of altered absorption and subsequent deposition of calcium in the skeletal pool.

Studies of the action of vitamin D in the rat.

1. Rats were given a rachitogenic diet for 28 days; some were dosed with vitamin D 2 or 3 days before they were killed and some received vitamin D throughout the experiment.2. Parathyroid leucine aminopeptidase was measured histochemically as an index of parathyroid activity and silver impregnation as a criterion of bone calcification.3. Calcium and phosphorus were estimated in bone ash and in serum. In rats killed 2 days after a single dose of vitamin D, increases were found in the Ca and P content of serum and bones and in epiphyseal Ca deposition. Parathyroid activity was decreased. In rats killed 3 days after a dose of vitamin D there was a marked increase in serum Ca and P but a less clear change in bone Ca and P although epiphyseal calcification was evident. The parathyroid activity was less than in rats dosed throughout the experiment.

Tryptic digestion and localization of calcium uptake and ATPase activity in fragments of sarcoplasmic reticulum

Fractions sedimented at 25,000 g for 50 minutes from homogenates of mouse hind leg muscles were prepared according to the procedure of Von Korff originally designed to obtain intact cardiac mitochondria (41). Negatively stained preparations of this fraction (fragmented sarcoplasmic reticulum) appear to be essentially free of contamination with mitochondrial fragments and myofilaments. Vesicles consisting of a globular head with a diameter of 0.1–0.2 μ and one or more tail-like portions with a roughly constant width of 350 and a varying length of 0.05–1 μ (most frequently 0.2 μ) predominate. The surface of the vesicle, both of the head and tail portion, is covered with particles approximately 40 in diameter and spaced 50–80 from each other, the center being 40–60 from the surface of the vesicular membrane. Ca deposits, in the presence of oxalate, were localized in the globular portion. ATPase activity, as judged electron microscopically from the deposition of lead phosphate, was also localized in the globular portion, very frequently at the junction with the tail. Digestion of vesicles with trypsin caused changes in both the Ca uptake and ATPase activity. With a trypsin-vesicular protein ratio of 1:25 to 1:100 a rapid drop in Ca uptake was accompanied by an increase in ATPase activity. With even lower (1:300) ratios, a rapid initial drop in Ca uptake was eventually followed by a slower phase of decrease; the ATPase in this case increased monotonically with an inflection point subsequent to the rapid drop in Ca2+ uptale. The digestion caused structural disruption of the vesicle, first in the tail, and eventually led to swelling of the globular portion and the appearance of areas devoid of regularly arranged particles on the membrane.

Effect of Varying Calcium Intake on the Parameters of Calcium Metabolism in the Rat

Kinetic tracer techniques providing the data for a compartmental analysis were supplemented with Ca balance measurements to obtain the rates of Ca absorption, endogenous fecal excretion, exchangeable space and accretion rates. The effect of low and high Ca diets was measured in terms of the above-measured parameters of skeletal metabolism in rats. Varying the Ca intake had no effect on the size of the compartments in the model used. Further, while the accretion rate of Ca into bone was not affected by the dietary Ca level, the rate of feedback of Ca from bone was quite sensitive to the level of Ca in the diet. The low Ca diet resulted in an appreciable increase in the resorption rate of Ca from bone and a very low rate of apposition of Ca into bone. However, the high Ca diet resulted in a minimal resorption rate (actually a negative rate) and a large increase in the net deposition of Ca in the skeleton. The use of a mathematical model proved to be a useful framework against which the effects of various Ca intakes could be measured quantitatively.

Staining methods for calcium--a comparison based on reactions shown by cardiac calcinosis in DBA mice.

Spontaneous calcification, which occurs frequently in hearts of mice of the DBA strain, has been used to compare effectiveness of calcium stains. Three fixatives (1) 80% ethanol, (2) 10% formalin and 3.5% acetic acid in 95% ethanol (modified Lavdowsky's) and (3) a 1:1 mixture of concentrated formalin and absolute ethanol were tested for their subsequent effects on staining. Stains for Ca included purpurin, von Kossa's, phthalocyanin, and glyoxal bis(2-hydroxyanil). These, with appropriate counterstaining, were compared among each other, with hematoxylin-eosin (H-E), and with the periodic acid-Schiff (PAS) reaction. Fixatives (1) and (3) did not affect results appreciably, but the von Kossa technic was unsatisfactory after (2). All stain demonstrated sites of Ca deposits, but purpurin and von Kossa's gave greatest sharpness of definition and were the simplest to perform. H-E and PAS with Alcian blue showed the same Ca sites in addition to details of the surrounding tissue.