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Roles of hydration, sodium, and chloride in regulation of canine mucociliary transport system.

To gain insight into the homeostatic mechanisms regulating airway ion/water fluxes and mucociliary transport, the canine tracheobronchial airway fluid was perturbed by deposition of hypo- and hyperosmotic aerosols for >1 h. Tracheal ciliary beat frequency (CBF) was measured by using heterodyne laser light scattering. Tracheal mucus velocity (TMV) and bronchial mucociliary clearance (BMC) were measured by using radioaerosols and nuclear imaging. Respiratory tract fluid output (RTFO) was collected by using a secretion-collecting endotracheal tube. In six dogs, CBF increased during water deposition in the airways to 180 +/- 30 mg/min and RTFO increased from 2.2 +/- 0.5 to 18.3 +/- 1.6 mg/min, accounting for <10% of the fluid deposition. TMV and BMC were unchanged. CBF, TMV, and BMC were markedly increased by inhalation of aerosolized 3.4 M NaCl. Aerosolized 0.85 M NaCl, in contrast, decreased BMC. In this case, RTFO represented 24% of aerosol deposition. Aerosolized 0.85 M choline chloride and 0.85 M sodium gluconate enhanced BMC and TMV concurrent with a decrease in CBF. RTFO of sodium gluconate studies exceeded 50% of aerosol deposition. Thus the airways appear to have transepithelial compensatory mechanisms that reduce the impact of a moderate increases in NaCl and hydration load, but when these responses cannot adequately respond because of the delivery of impermeable ions or very high tonicity, removal of the challenges are affected by a stimulation of mucociliary transport.

Aerosols↗

Taste-mixture suppression: functional dissection of cellular and paracellular origins.

1. Chorda tympani (CT) nerve responses were recorded during simultaneous current and voltage clamping of the lingual receptive-field epithelium to examine the role of field potential in taste mixture suppression between sodium gluconate (NaG) and potassium gluconate (KG). 2. Under zero current-clamp conditions, CT responses to 100 mM NaG were suppressed by 63% when presented in mixture with 250 mM KG. At this concentration, KG alone elicited no measurable neural activity, but produced a large submucosal-positive field potential. 3. When CT responses to 100 mM NaG were obtained with voltage clamp at the zero-current clamp field potential of the NaG/KG mixture, they were suppressed by only 30% relative to NaG responses under zero-current clamp. Similarly, CT responses to the mixture of NaG and KG measured while voltage was clamped at the field potential of NaG alone were slightly elevated, but not to the magnitude of zero-current clamp responses to NaG. Therefore field potential-mediated suppression of CT responses to NaG accounts for only a part of the total mixture suppression between NaG and KG. 4. Analysis of the voltage dependence of CT responses to NaG indicated that the moderate field potential increase (8.9 mV) caused by the presence of KG in the mixture equates to a 43% increase in the apparent Km for NaG, from 110 to 157 mM. Use of this effective Km obviated the effect of field potential on CT responses to the NaG/KG mixture and permitted kinetic analysis of K+ blockade of Na+ responses. These analyses suggested that K ions block Na+ movement through apical Na+ channels in a voltage-independent manner with an apparent Ko of 405 mM. Importantly, direct inhibition of Na+ transduction by K+ can account for the part of mixture suppression not mediated by field potential. 5. These experiments reveal that mixture suppression between NaG and KG is derived from two distinct sources. Field potential, triggered largely by the limited mobility of both K+ and Na+ through taste bud tight junctions, globally modulates Na+ transduction. In addition, at the level of the apical Na+ channel, K ions directly block movement of depolarizing Na+ across taste receptor apical membranes.

Animals↗

Intrarenal site of action of calcium on renin secretion in dogs.

We studied the effects of intrarenal calcium infusion on renin secretion in sodium-depleted dogs in an attempt to elucidate the major site of calcium-induced inhibition of renin release. Both calcium chloride and calcium gluconate reduced renal blood flow and renin secretion while renal perfusion pressure was unchanged. These data indicate that calcium inhibition of renin secretion did not occur primarily at the renal vascular receptor; decreased renal blood flow is usually associated with increased renin secretion. Calcium chloride infusion increased urinary chloride excretion without affecting sodium excretion, and calcium gluconate failed to increase either sodium or chloride excretion. Also, the filtered loads of sodium and chloride were unchanged during the calcium infusions. These results give no indication that calcium inhibited renin secretion by increasing the sodium or chloride load at the macula densa. The effects of intrarenal calcium infusion on renin release were also assessed in dogs with a nonfiltering kidney in which renal tubular mechanisms could not influence renin secretion. The observation that calcium still suppressed renin release in these dogs provides additional evidence that the the major effect of calcium involved nontubular mechanisms. Thus, it appears likely that calcium acted directly on the juxtaglomerular cells to inhibit renin secretion.

Animals↗

Comparative clinical evaluation of two salts of quinidine using 24-hour Holter monitor recordings.

In 13 patients admitted to a coronary care unit, antiarrhythmic efficacy and safety of quinidine gluconate in a nonmatrix formulation were compared to placebo and quinidine sulfate. Both quinidine preparations were effective in reducing the number and severity of ventricular and supraventricular extrasystoles when compared to placebo. Except for a few cases in which quinidine gluconate was significantly more effective, both quinidine preparations appear to have similar clinical antiarrhythmic efficacy and duration of action. A 24-hour Holter ECG monitoring device and electrocardioscanner were utilized in this research. No major unexpected adverse effects were observed.

Aged↗

Aluminum in parenteral nutrition solution--sources and possible alternatives.

The extent of aluminum (Al) contamination in parenteral nutrition (PN) solutions for infants is not known. Aluminum was measured in 136 samples from various commercially available components that are used with PN. Results showed Al content varied widely among different components. The same chemical may have a different Al content depending on the manufacturer. However, Al contents were similar among lots from the same manufacturer for the same chemical. Aluminum contamination was arbitrarily classified as high (greater than 500 micrograms Al/liter), intermediate (51-500 micrograms Al/liter) or low (less than or equal to 50 micrograms Al/liter). The high group included most calcium and phosphorus containing salts, 1 multivitamin preparation, folic acid, ascorbic acid and concentrated (25%) albumin. The intermediate group included sodium lactate, potassium phosphates, zinc and chromium chloride, multitrace metal preparation, and 5% plasma protein. The low group included amino acids, sterile water and dextrose water, chloride salts of sodium, potassium, calcium, copper and chromium, sodium phosphates, magnesium sulphate, zinc sulphate, vitamin B12, vitamin K1, 1 multivitamin preparation, soybean oil emulsion and heparinized (2 U/ml) saline. PN solutions made from high Al components may contain up to 300 micrograms Al/liter. Calcium gluconate contributed greater than 80% of the total Al load from PN. Lowering of Al content in calcium gluconate in addition to use of specific low Al components offers the opportunity to significantly lower the Al concentration of the final PN solution and theoretically may achieve an Al content as low as 12 micrograms/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminum↗

Glycero- vs glucose-phosphate in parenteral nutrition of premature infants: a comparative in vitro evaluation of calcium/phosphorus compatibility.

In total parenteral nutrition (TPN) of premature infants, glycero- and glucose-phosphate have been recommended, and clinically used, because of their considerable compatibility with calcium. However, a systematic comparative in vitro assessment of the therapeutic potential and safety of these substances has not yet been provided. We investigated the stability of TPN solutions containing calcium-gluconate and glycero- or glucose-phosphate in high concentrations. Evaluation was performed by visual inspection, absorptiometry, light microscopy, measurement of pH, and determination of calcium concentration before and after microfiltration. Even under circumstances promoting precipitation of calcium and phosphate--such as body temperature, relatively high pH, and concentrations of calcium and phosphorus exceeding those necessary to provide intrauterine accretion rates, all but one of the examined TPN admixtures remained stable. Our data suggest that the use of glycero-phosphate, and particularly glucose-phosphate, together with calcium-gluconate, is an uncomplicated and safe procedure to administer simultaneously high amounts of calcium and phosphorus in TPN of premature infants.

Calcium↗

Regulation of calciotropic hormones in vivo in the New Zealand white rabbit.

Serum levels of ionized calcium, 25-hydroxyvitamin D (25OHD), and 1,25-dihydroxyvitamin D[1,25-(OH)2D], intact immunoreactive PTH and calcitonin were measured in the laboratory rabbit to evaluate the role of these calciotropic hormones in calcium homeostasis in this species. We confirm the finding of previous researchers that the resting serum ionized and total calcium concentrations are elevated in rabbits compared to those in other species (ionized calcium, 1.70 +/- 0.13 mmol/liter; total calcium, 3.23 +/- 0.25 mmol/liter). The serum calcium concentrations in animals maintained on a breeding farm or in the laboratory did not differ significantly despite nearly 3-fold higher levels of vitamin D in the feed at the farm, which were associated with 3- to 4-fold higher concentrations of 25OHD and 1,25-(OH)2D. Baseline intact PTH levels for the farm and laboratory populations also did not differ significantly and averaged 69.4 +/- 43.6 human pgeq/ml (laboratory animals, 52.1 +/- 28.4; breeding farm animals, 86.0 +/- 49.5 human pgeq/ml). Infusions of calcium gluconate or EDTA for 15 min into anesthetized animals in the laboratory induced dramatic reciprocal changes in the measured circulating levels of PTH. Calcium gluconate infusions (190-300 nmol/g BW) produced 50-85% increases in serum ionized calcium, which were accompanied by 74-91% decreases in PTH levels (from 68.8 +/- 29.2 at time zero to 10.1 +/- 3.1 human pgeq/ml at 15 min) as well as 7-fold increases in calcitonin levels. EDTA infusions (14-120 nmol/g BW) reduced serum ionized calcium by 9-49%, while PTH levels increased by 68-560% (from 61.4 +/- 32.3 at time zero to a maximum of 138 +/- 48.6 human pgeq/ml at 3 min). During the EDTA infusion, the PTH response was variable after 3 min despite further decreases in ionized Ca2+, indicating either exhaustion of PTH reserves or regulation of the secretory response by some parameter other than ionized calcium concentration per se. Thus, the rabbit appears to defend its serum ionized calcium concentration against hypo- and hypercalcemia by rapid changes in PTH secretion and calcitonin. Unlike other mammalian species, however, the changes in PTH occur at relatively high levels of calcium, suggesting that the parathyroid gland of the rabbit is reset to respond to changes in ionized Ca2+ within the physiological range in that species. The relative insensitivity of the rabbit parathyroid to extracellular calcium is analogous to that observed in primary hyperparathyroidism and may be a useful model to study the control of normal and abnormal PTH secretion.

Animals↗

Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans.

We employed variable rates of infusion of EDTA or calcium gluconate to examine the relationships between the concentration and rate of change of serum total and plasma ionized calcium and serum immunoreactive intact PTH concentrations in normal subjects. Use of a sensitive immunoradiometric assay specific for PTH-(1-84) made it possible to determine the full range of PTH responses to perturbations in the extracellular calcium concentration. By progressively increasing the rate of administration of calcium gluconate or EDTA during rapid or slow infusions, linear rates of change in the serum calcium concentration were achieved in eight men. The slopes were 2-fold greater during the rapid infusions. Both the calcium infusions decreased serum PTH-(1-84) levels from a mean of 23.2 +/- 2.0 (+/- SE) to 6.4 +/- 1.0 and 5.6 +/- 1.0 ng/L for the rapid and slow infusions, respectively, with no obvious rate dependence. The rapid or slow EDTA infusions increased serum PTH levels to the same maximal extent (95.0 +/- 20.2 and 99.9 +/- 14.5 ng/L, respectively), also with no significant rate dependence. Thus, there was no effect of the rate of change of calcium on the PTH response, which was reflected in similar inverse sigmoidal relationships between PTH and serum total and plasma ionized calcium when the data for the slow and rapid infusions were pooled separately. Similar sigmoidal curves were found in normal women. These data suggest the feasibility of using calcium and EDTA infusions combined with an intact PTH assay to define the relationships between circulating levels of PTH-(1-84) and calcium in states of normal and deranged parathyroid physiology.

Adult↗

A new method for in vivo analysis of parathyroid hormone-calcium set point in mice.

Although methods for measuring the parathyroid hormone (PTH)-calcium set point in vivo in humans and large animals exist, translating such methods to the increasingly important mouse model poses considerable challenges. We also found that manipulation of dietary calcium does not yield sufficiently high or low serum calcium levels to achieve the minimum and maximum PTH levels needed for set point estimations. Therefore, we developed a new method for in vivo evaluation of the relative set point in mice. Intraperitoneal injection of calcium gluconate caused progressive increases in serum calcium over 120 minutes, with corresponding decreases in serum PTH levels. Intraperitoneal injection of Na2-EGTA caused a nadir of serum calcium at 30 minutes and a corresponding peak value of serum PTH. The lowest and highest serum calcium concentrations achieved were 6.7 mg/dl and 10.0 mg/dl, respectively. Linear regression analyses indicated high correlation coefficients (serum calcium vs. serum PTH; r = 0.969). To obtain the additional data points needed for set point estimation, blood was collected 30 minutes after intraperitoneal injection by tail nicking from three mice once a day, on 8 consecutive days, using multiple doses of calcium gluconate or Na2-EGTA. The lowest and highest serum calcium concentrations achieved were 5.0 mg/dl and 11.4 mg/dl. Maximum and minimum PTH levels were indeed observed, and a sigmoidal curve with a set point of 7.8 mg/dl was readily generated by the four-parameter model that was fit using a nonlinear mixed effects statistical approach. This protocol for in vivo set point analysis should be applicable in the future study of multiple genetically engineered and pharmacologically treated mouse models.

Animals↗

Sorbitol can be produced not only chemically but also biotechnologically.

Sorbitol, a polyol found in many fruits, is attracting increasing industrial interest as a sweetener, humectant, texturizer, and softener. It is principally produced by chemical means. The bacterium Zymomonas mobilis is able to produce sorbitol together with gluconic acid from fructose and glucose, respectively. This is possible in a one-step reaction via the enzyme glucose-fructose oxidoreductase, so far only known from Z. mobilis. The possibilities for the production of sorbitol by Z. mobilis are discussed also under the aspect of an industrial process and compared with the current chemical as well as other microbiologic processes. The production process by Z. mobilis shows economic possibilities for certain countries, such as Brazil, considering only the products sorbitol and ethanol as an important byproduct. For the other byproduct, gluconic acid, further studies for its partial substitution must be conducted.

Biotechnology↗

Normal growth rate in rats is recovered after a period of zinc deficiency by restoration of zinc supply by means of a zinc-fortified Petit Suisse cheese.

Fortification of a Petit Suisse cheese with zinc sulfate and zinc gluconate stabilized with glycine was used as a tool to overcome zinc-deficiency effects on total-body growth and skeletal growth. Animals were divided in 4 groups of 10 rats: basal (B), control (C), depletion-repletion 1 (DR1), and depletion-repletion 2 (DR2). These four groups were fed with four diets: basal (2 ppm Zn), control (30 ppm Zn), DR1, and DR2; they received a basal diet for 14 d and a control diet for the other 14 d of the experiment, using zinc sulfate for DR1 and zinc gluconate stabilized with glycine for DR2. After 28 d of the experiment, total-body weight and weight gain of the control and DR1 and DR2 animals were not statistically different (p<0.05), Femur weight and femur zinc content of DR1 and DR2 did not achieve the values of control animals (p<0.05), but they were higher than that of basal animals. Our results show that restoration of dietary zinc levels by means of food fortification normalized weight gain, as an indicator of total-body growth, and presented a trend to normalize bone weight, as a marker of skeletal growth, in young rats and independently of the zinc source used.

Animals↗

Effect of acute hypercalcaemia on glucose tolerance and insulin release in human beings.

Insulin secretion in response to an oral glucose challenge during acute hypercalcaemia was studied. Oral glucose tolerance tests (OGTT) were performed in 12 non-diabetic, non-obese human volunteers, aged 20-28 years. Blood samples were collected for calcium, glucose and insulin determination. The next day the same volunteers received a 4 h infusion of calcium gluconate (15 mg/kg/4 h) and were administered glucose 1 h after starting the infusion. Serum calcium, glucose and insulin concentrations were measured again. Infusion of calcium gluconate resulted in an increase in serum calcium concentration of 5 mg/100 ml over 4 h. During these infusions no significant changes in glucose concentrations were noted. On the other hand, the total mean insulin concentration, expressed as the area under the 3 h glucose tolerance curve, and the insulin peak at 30 min were significantly increased during hypercalcaemia (P < 0.001). These data indicate that acute hypercalcaemia does not affect carbohydrate tolerance but increases insulin secretion in human beings.

Adult↗

In vivo effect of endothelin-1 on plasma calcium and parathyroid hormone concentrations.

We have previously reported an in vitro inhibitory effect of endothelin-1 (ET-1) on parathyroid hormone (PTH) secretion. In the present experiment, ET-1 was infused into rabbits to study the in vivo effect of ET-1 on the changes in calcium, magnesium, PTH and calcitonin concentrations. Femoral arteries and veins of anesthetized male rabbits were cannulated to monitor vital signs, blood sampling and infusion of the agents being studied. Infusion of ET-1 (1, 5, 10 and 20 ng/kg per min) induced a dose-dependent decline in plasma ionized calcium concentrations from 6.68+/-0.26 to 5.50+/-0.46 mg/dl (P<0.05) and a decrease in calcitonin concentrations from 48.6+/-6.5 to 32.5+/-4.7 pg/ml. PTH concentrations increased from 58.3+/-10.2 to 159.4+/-22.1 pg/ml. In a separate experiment, calcium gluconate solution was simultaneously infused to keep calcium concentrations steady, thereby proving a calcium 'clamp'. In normal calcium concentration, ET-1 infusion gradually decreased PTH concentrations from 71.4+/-8.6 to 38.0+/-6.2 pg/ml. We further infused sodium citrate solution to decrease the calcium concentration (2.0 mg/dl less) and calcium gluconate solution was infused to keep calcium concentrations steadily less than normal. PTH concentrations were initially stimulated by the induction of hypocalcemia (68.1+/-11.2 to 135.6+/-8.5 pg/ml), but decreased by ET-1 infusion (135.6+/-8.5 to 85.1+/-15.2 pg/ml). Plasma magnesium concentrations did not change significantly throughout the entire study and calcitonin concentrations were not significantly changed during the calcium clamp studies. Serum phosphate and 1,25-(OH)(2) vitamin D(3) concentrations were also measured, but they also did not change significantly. In conclusion, ET-1 exhibited an in vivo acute hypocalcemic action, independent of calcitonin. It also directly decreased PTH secretion if serum calcium concentrations were kept steady. The above findings are consistent with the results of our previous in vitro experiment.

Animals↗

The large intestine compensates for insufficient calcium absorption in the small intestine in rats.

We previously demonstrated that the large intestine compensated for decreased calcium (Ca) absorption caused by renal failure in rats fed a highly fermentable dietary fiber. In this study, we examined whether the large intestine compensated for insufficient Ca absorption in the rat small intestine without ingestion of a fermentable dietary fiber. Rats were fed one of four test diets containing either insoluble (carbonate) or soluble (gluconate, lactate, or citrate) Ca sources. The dietary Ca level was 2.0 g/kg, which is lower than the minimum requirement for rats (3.0 g/kg), to conduct the present study under a condition in which rats can maximally absorb Ca. To prevent Ca absorption in the small intestine, we replaced a primary phosphate (KH2PO4) with secondary phosphates (K2HPO4 and Na2HPO4) in diets. The apparent Ca absorption in the small intestine was estimated by adding chromic oxide (Cr2O3) as an insoluble and an unabsorbed marker to test diets and by measuring the ratio of Ca:Cr in the cecal content. The apparent Ca absorption in the whole intestine was estimated by the intake and fecal excretion of Ca. The apparent Ca absorption in the small intestine was significantly lower from the Ca carbonate diet than from the Ca gluconate, lactate, or citrate diets. The apparent Ca absorption in the whole intestine was not significantly different among the four groups, and the values were similar to the absorption rates in the small intestines of rats fed diets containing soluble Ca sources. These results show the following: (a) In rats fed 0.2% Ca diets containing soluble Ca salts, Ca is mostly absorbed in the small intestine, even in secondary phosphate intakes; (b) In contrast, in rats fed a 0.2% Ca diet containing an insoluble Ca salt (carbonate), Ca is not sufficiently absorbed in the small intestine. However, the large intestine compensates for the small intestinal Ca absorption decreased by dietary secondary phosphates.

Animals↗

Inhibitory effects of chloride on the activation of caspase-1, IL-1beta secretion, and cytolysis by the P2X7 receptor.

The P2X7 receptor (P2X7R) is an ATP-gated cation channel that activates caspase-1 leading to the maturation and secretion of IL-1beta. Because previous studies indicated that extracellular Cl- exerts a negative allosteric effect on ATP-gating of P2X7R channels, we tested whether Cl- attenuates the P2X7R-->caspase-1-->IL-1beta signaling cascade in murine and human macrophages. In Bac1 murine macrophages, substitution of extracellular Cl- with gluconate produced a 10-fold increase in the rate and extent of ATP-induced IL-1beta processing and secretion, while reducing the EC50 for ATP by 5-fold. Replacement of Cl- with gluconate also increased the potency of ATP as an inducer of mature IL-1beta secretion in primary mouse bone marrow-derived macrophages and in THP-1 human monocytes/macrophages. Our observations were consistent with actions of Cl- at three levels: 1) a negative allosteric effect of Cl-, which limits the ability of ATP to gate the P2X7R-mediated cation fluxes that trigger caspase-1 activation; 2) an intracellular accumulation of Cl- via nonselective pores induced by P2X7R with consequential repression of caspase-1-mediated processing of IL-1beta; and 3) a facilitative effect of Cl- substitution on the cytolytic release of unprocessed pro-IL-1beta that occurs with sustained activation of P2X7R. This cytolysis was repressed by the cytoprotectant glycine, permitting dissociation of P2X7R-regulated secretion of mature IL-1beta from the lytic release of pro-IL-1beta. These results suggest that under physiological conditions P2X7R are maintained in a conformationally restrained state that limits channel gating and coupling of the receptor to signaling pathways that regulate caspase-1.

Animals↗

Efficacy of combined immunostimulation and chemotherapy in experimental visceral Leishmaniasis.

A regimen of combined immunostimulation and chemotherapy for the elimination of Leishmania donovani amastigotes was evaluated. An in vitro experimental model utilized cultured peritoneal macrophages from C57B1/6 mice infected with L. donovani tissue forms. Partial or complete activation of macrophages as judged by killing of tumor cells significantly enhanced the efficacy of sodium antimony gluconate (Pentostam). The quantity of drug required for elimination of parasites from immunostimulated cells was considerably lower than that required to achieve comparable amastigote killing in thioglycolate-elicited macrophages. In contrast, amphotericin B cleared infected cells of amastigotes at comparable drug levels when tested with immunostimulated and unstimulated macrophages. Several drugs tested inhibited the conversion of amastigotes to promastigotes in vitro but were ineffective in killing of intracellular tissue forms. Allopurinol and difluoromethylornithine (DMFO) blocked amastigote conversion significantly. These drugs at high concentrations, however, exerted only minimal toxicity for amastigotes residing within macrophages. Efficacy of combined therapy was also demonstrated in vivo. Immunoenhancement of L. donovani-infected mice with Corynebacterium parvum vaccine combined with a regimen of sodium antimony gluconate was significantly more effective than was immunotherapy or drug therapy alone.

Amphotericin B↗

Severe calcinosis cutis associated with treatment of hypoparathyroidism in a dog.

A 6-month-old, female border collie was referred for evaluation of hypocalcemia, hyperphosphatemia, fever, and painful ventral abdominal skin. She had recently been treated intravenously and subcutaneously (SC) with a diluted 10% calcium gluconate solution. The medical evaluation supported the diagnosis of primary hypoparathyroidism, but the subsequent hospital course was complicated by severe calcinosis cutis, which caused extensive skin necrosis and marked debilitation. This patient illustrates that administration of a calcium gluconate solution SC can be associated with extensive morbidity when administered to hyperphosphatemic patients.

Abdomen↗

Primary hypoparathyroidism in a cat complicated by suspect iatrogenic calcinosis cutis.

An adult male cat presented to the Ontario Veterinary College after having a seizure at the humane society. The cat was diagnosed with primary hypoparathyroidism and was treated with calcium and vitamin D3 supplementation. Calcium supplementation included subcutaneous (SC) administration of diluted calcium gluconate according to protocols described in recent literature. Seven days after SC calcium administration, firm SC masses developed in the intrascapular area where the injections were given and in the dependent axillary area. These areas became raised, formed crusts, necrosed, and left scars. Although not histopathologically confirmed, the author postulates that this was a direct reaction to SC calcium gluconate administration.

Animals↗