Extracellular iCa concentrations are regulated by a complex homeostatic system that includes 3 hormones (PTH, calcitonin, and 1,25-dihydroxyvitamin D3); 3 body systems (the bones, intestines, and kidneys); and CaRs in the chief cells of the parathyroid gland, C cells of the thyroid gland, and renal tubular cells.1 A homeostatic system for extracellular iMg is not clearly defined, and extracellular concentrations of iMg are primarily controlled by gastrointestinal absorption, renal reabsorption, and to a lesser degree, hormones such as PTH and insulin.2,3
Although the main role of PTH is to increase reabsorption of iCa in the kidneys and osteoclastic resorption in bones, renal reabsorption of iCa is also regulated by PTH-independent mechanisms, primarily by the CaR that affects transepithelial transport of iCa and iMg in the TAL and distal convoluted tubules.1,4,5 Activation of the CaR in the TAL inhibits the sodium-potassiumchloride cotransporter, which is essential for paracellular reabsorption of iCa and iMg and transcellular transport of sodium, potassium, and chloride.1
Electrolyte disturbances are common in critically ill horses. Hypocalcemia and hypomagnesemia have been reported in sick human and veterinary patients.6–9 Low serum concentrations of iCa and iMg are common laboratory abnormalities in critically ill horses, especially animals with gastrointestinal tract diseases.7,9 Hypocalcemia in horses is associated with ileus, seizures, dyspnea, tetany, tremors, cardiac arrhythmias, and death.10 Hypomagnesemia has been associated with increased fatalities in human and equine patients.7,11
Although limited information is available on hypocalcemia and hypomagnesemia in sick horses, less is known about hypercalcemia in horses, especially the effects of increased extracellular iCa on other electrolytes. Hypercalcemia is a common finding in horses with pathologic conditions, such as chronic renal failure, neoplasia, hyperparathyroidism, and vitamin D intoxication.10 In humans and laboratory animals, it has been reported that hypercalcemia results in polyuria, urinary concentration defects, and water and sodium wasting.1,12–15 Clinical experience suggests that there may be a similar phenomenon in hypercalcemic horses; however, to our knowledge, no information on the effects of hypercalcemia on serum and urinary electrolytes in equids is available. In another study16 conducted by our laboratory group, we reported that increased extracellular iCa concentrations increase CaR activation and CaR mRNA expression, which decreases PTH mRNA expression and PTH secretion in equine parathyroid cells.
Because iCa can potentially affect other electrolytes and because there is limited information on renal iCa regulation in horses, the objective of the study reported here was to evaluate the effects of experimentally induced hypercalcemia on serum concentrations of electrolytes and their urinary excretion in healthy horses, with particular emphasis on iMg. On the basis of our current knowledge of CaR function, we speculated that hypercalcemia in horses would activate CaRs in chief cells of the parathyroid gland to decrease PTH secretion and in renal tubular cells to decrease renal reabsorption of iCa, iMg, potassium, sodium, and chloride, which would thus increase urinary excretion of electrolytes and urine production. Two calcium salts that are used in human and veterinary medicine to treat patients with hypocalcemia (CaGlu and CaCl2) were used to induce hypercalcemia. In addition, solutions of sodium gluconate and dextrose were infused to separate the effects of calcium from those of gluconate.
Materials and Methods
Animals—Twenty-one healthy mares (3 to 12 years old; mean ± SD, 7.7 ± 3.0 years) that weighed 465 to 565 kg (mean, 510 ± 18 kg) were selected from a university teaching herd. All horses were in good body condition, were fed the same diet of grass and grass hay (0.5% calcium and 0.25% phosphorus) and alfalfa hay (1.4% calcium and 0.25% phosphorus), had no history of illness, and had received no treatments for 1 month prior to the study. To ensure horses were healthy, a complete physical examination was performed. Values for a CBC and serum biochemical analysis; serum concentrations of tCa, iCa, tMg, iMg, phosphate, PTH, and insulin; plasma concentrations of fibrinogen; results of blood gas analyses and urinalysis; and FCa, FMg, FNa, FPO4, FK, and FCl were within the respective reference ranges for all horses. The University Institutional Laboratory Animal Care and Use Committee approved this study, and horses were treated in accordance with National Institutes of Health Institutional Animal Care and Use guidelines.
Experimental procedures—Horses received no food or water during the experiments. Only mares were used in the study to facilitate urine collection. To our knowledge, there is no published information to suggest differences between male and female horses for the variables evaluated in the study.
Horses were assigned to 5 experimental protocols. Six horses were assigned to protocol 1 (hypercalcemia induced by administration of CaGlu), and 6 horses were assigned to protocol 2 (hypercalcemia induced by administration of CaCl2). In contrast, 3 horses were assigned to protocol 3 (infusion with dextrose solution), 3 horses were assigned to protocol 4 (infusion with NaGlu), and 3 horses were assigned to protocol 5 (control; infusion with saline [0.9% NaCl] solution). All solutions were adjusted to pH 7.4. For accurate volume delivery, solutions were administered by use of IV fluid infusion pumps.a
The goal of the calcium infusions was to increase serum iCa concentrations from baseline values (mean ± SD, 6.6 ± 0.2 mg/dL; range, 6.0 to 7.0 mg/dL) to 10.0 mg/dL (ie, 2.5 mmol/L) to induce maximal CaR activation in the parathyroid chief cells and renal tubular cells. Because the CaGlu solution contained 21% gluconate, which is an oxidation product of glucose, equivalent solutions of dextrose and NaGlu were infused with the goal of separating the effects of extracellular iCa on serum and urinary electrolytes from effects attributable to gluconate. The authors were not certain as to whether the administration of gluconate salts would result in hyperglycemia or stimulate insulin release, which can alter the serum concentrations of electrolytes such as magnesium, potassium, and phosphate. In addition, for physiologic pH conditions, gluconate salts dissociate to gluconic acid, which is an organic anion that can chelate various metals, including calcium and magnesium.17 Thus, there was the possibility of reducing extracellular concentrations of these metals.
Limited information is available on the parenteral use of NaGlu18–21; however, CaGlu is widely used for IV administration in human and veterinary medicine. On the basis of low stability constants (chelating effect) of gluconic acid for calcium and magnesium (1.2 and 0.7, respectively), compared with the stability constants for EDTA (10.7 for calcium and 8.8 for magnesium), determined by the National Institute of Standards and Technology,17 which our laboratory group has used extensively in horses,22 and from toxicologic information obtained after parenteral administration in other species,18-21,23 we predicted that there would be no adverse effects after parenteral administration of NaGlu at the dosages used in our experiments. To monitor for potential adverse effects during infusions, heart rate, respiratory rate, capillary refill time, and an ECG (lead II) were evaluated at intervals of 15 to 20 minutes during the experiments.
Induction of hypercalcemia by administration of CaGlu (protocol 1)—Hypercalcemia was induced in 6 healthy mares by administration of a 23% CaGlu solutionb that contained 21.4 mg of elemental calcium/mL. To induce hypercalcemia in a stepwise manner, horses were infused for 120 minutes. There was an initial elemental calcium rate of 4 mg/kg/h with increases of 2 mg/kg/h every 15 minutes to achieve a final rate of 16 mg/kg/h for the final 30 minutes of each infusion. Total volume infused ranged from 460 to 530 mL. Time of initiation of infusion was designated as time 0. Elemental calcium infusion rates of 4 to 8 mg/kg/h are within the rates that we routinely use in critically ill hypocalcemic horses admitted to The Ohio State University Equine Intensive Care Unit.
Induction of hypercalcemia by administration of CaCl2 (protocol 2)—Hypercalcemia was induced in 6 healthy mares by administration of a 10% CaCl2c solution that contained 27.2 mg of elemental calcium/mL. The infusion was administered in a stepwise manner similar to that for protocol 1. Total volume of 10% CaCl2 infused ranged from 380 to 420 mL.
Infusion of dextrose solution (protocol 3)—Three healthy mares were infused with a 21% dextrose solution.d Similar to the infusion for protocol 1, there were stepwise increases every 15 minutes during the 120-minute infusion period. This represented a dextrose infusion rate that ranged from 20 to 160 mg/kg/h at the end of the infusion. Total volume infused ranged from 435 to 520 mL.
Infusion of NaGlu (protocol 4)—Three healthy mares were infused with a 21% solution of NaGlue with stepwise increases every 15 minutes for 120 minutes, similar to other protocols. The rate was equivalent to the gluconate rate in the 23% CaGlu infusion (ie, an increase in NaGlu infusion rate from 20 to 160 mg/kg/h at the end of the 120-minute infusion period). Total volume of NaGlu solution infused ranged from 450 to 510 mL.
Infusion of saline solution (protocol 5)—Three control horses were infused with an equivalent volume of saline solutionf at a rate of 0.5 mL/kg/h for 120 minutes. Total volume infused was 470 to 510 mL.
Infusions and sample collection—A catheterg was aseptically inserted in each jugular vein of each horse. The catheter in the left jugular vein was used for the infusion of CaGlu, CaCl2, dextrose, NaGlu, or saline solutions, whereas the catheter in the right jugular vein was used for collection of blood samples.
Blood samples were collected every 10 minutes from 0 (baseline) to 300 minutes, and additional samples were collected at 300 and 360 minutes. Venous blood samples were collected in tubes with no additives, allowed to clot for 1 hour, and then centrifuged at 1,000 X g for 5 minutes at 4°C. Samples collected at 10-minute intervals and at 360 minutes were used to measure iCa, iMg, sodium, potassium, and chloride concentrations. Blood samples used for measurement of serum iCa and iMg concentrations were collected by use of anaerobic conditions. Serum concentrations of tCa, tMg, phosphate, glucose, insulin, PTH, bicarbonate, and creatinine and pH were measured in samples collected at 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, and 360 minutes. Serum samples for biochemical analysis and measurement of creatinine, glucose, tCa, tMg, iCa, iMg, phosphate, and bicarbonate concentrations were processed immediately, whereas samples used to measure PTH and insulin concentrations were stored at −80°C until subsequent batch analysis.
Venous blood gas determinations were conducted on samples obtained by use of anaerobic conditions every 30 minutes. These samples were processed immediately after collection.
Urine samples were collected by inserting an indwelling 28-F Foley catheter into the bladder of 4 horses for protocols 1 and 2 and each of the 3 horses for protocols 3, 4, and 5. Urine was allowed to flow continuously into individual collection containers that were emptied after the volume was recorded at the end of each collection period. Urine samples were collected 30 minutes before (baseline) initiation of infusions and at 30-minute intervals thereafter until 360 minutes after the initiation of infusions. Urine samples to determine electrolyte and creatinine concentrations were processed immediately. Urine output, specific gravity, osmolality, volume, and pH were also determined.
Urine samples collected at 30-minute intervals were also used to calculate FCa, FMg, FNa, FPO4, FK, and FCl. Urinary fractional excretion of electrolytes was calculated by use of the following equation:
where Fx is the urinary fractional excretion of electrolyte x, Ux is the urinary concentration of electrolyte x, Sx is the serum concentration of electrolyte x, Ucr is the urinary creatinine concentration, and Scr is the serum creatinine concentration. Therefore, the results were expressed as a fraction (percentage) of the urinary excretion of creatinine. Fractional excretion of electrolytes was calculated instead of urinary clearance, which assesses glomerular filtered loads of electrolytes, because we were interested in the urinary excretions at the end of each 30-minute time period.
Laboratory analysis—Hemograms were created by use of an automated system.h For specific measurements of urine concentrations of tCa, tMg, and phosphate, a 1:10 volume of 6N HCl was added to each urine sample to dissolve crystals and prevent crystal formation. Variables for serum biochemical analysis; serum concentrations of creatinine, glucose, tCa, phosphate, sodium, potassium, and chloride; and urine concentrations of creatinine, tCa, sodium, potassium, chloride, and phosphate were measured by use of an automated analyzer.i Serum and urine concentrations of tMg were determined by use of a chemistry system.j Serum concentrations of iCa and iMg were measured by use of calcium- or magnesium-selective electrodes.k Serum concentrations of PTH were determined by use of an immunochemiluminometric assay for human intact PTHl that has been validated for use in samples obtained from horses.9 Serum concentrations of insulin were determined by use of a human-specific insulin radioimmunoassaym that has been used on samples obtained from horses.24 Plasma fibrinogen concentrations were determined by use of a nephelometric analyzer.n Blood gases were measured by use of a blood gas analysis system.o
Statistical analysis—Results were expressed as mean ± SD. Normality was determined by visual inspection of normal probability plots and by use of the Shapiro-Wilk goodness-of-fit test. For correlation between variables, the Pearson product moment test was used for normally distributed variables and the Spearman rank test for variables that were not normally distributed. Comparisons between 2 groups were made by use of the t test or Mann-Whitney rank test, depending on normality of the data. A 1-way repeated-measures ANOVA and multiple comparisons with the Dunnett test were used to compare values at various time points with the value for the baseline (time 0) value. When variables were not normally distributed, the Friedman 1-way ANOVA for repeated measures was used, and multiple comparisons with time 0 were made by use of the Dunn test. Statistical analyses were performed by use of statistical software,p,q and graphs were generated by use of a plotting program.r Values of P < 0.05 were considered significant.
Results
Horses tolerated the infusions well. No abnormalities were detected during physical evaluations in any of the horses during the experiments.
The induction of hypercalcemia resulted in changes in serum electrolyte concentrations and urinary fractional excretions. The most consistent findings with hypercalcemia induced by administration of CaGlu or CaCl2 were the development of hypomagnesemia, hypokalemia, and hyperphosphatemia. The infusion of dextrose solution and NaGlu also resulted in a mild decrease in serum iMg and potassium concentrations; however, no significant changes in other electrolyte concentrations were detected (Table 1). No changes in serum concentrations of electrolytes were detected in horses infused with saline solution.
Mean ± SD serum concentrations of electrolytes and other substances measured in healthy horses infused with CaGlu, CaCl2, dextrose, and NaGlu solutions.
| Variable | CaGlu (protocol 1; n = 6) | CaCl2 (protocol 2; n = 6) | Dextrose (protocol 3; n = 3) | NaGlu (protocol 4; n = 3) | ||||
|---|---|---|---|---|---|---|---|---|
| Baseline | Infusion | Baseline | Infusion | Baseline | Infusion | Baseline | Infusion | |
| iCa (mg/dL) | 6.6 ± 0.3 | 9.7 ± 0.7* (120) | 6.4 ± 0.2 | 10.2 ± 0.3* (120) | 6.4 ± 0.3 | 6.2 ± 0.2 (140) | 6.6 ± 0.3 | 6.3 ± 0.2* (180) |
| tCa (mg/dL) | 12.3 ± 0.4 | 21.4 ± 1.0* (120) | 12.0 ± 0.5 | 21.8 ± 0.8* (120) | 12.1 ± 0.4 | 11.8 ± 0.6 (140) | 12.2 ± 0.4 | 11.8 ± 0.6 (180) |
| iMg (mmol/L) | 0.52 ± 0.04 | 0.33 ± 0.05* (140) | 0.52 ± 0.03 | 0.38 ± 0.04* (140) | 0.50 ± 0.04 | 0.42 ± 0.04* (130) | 0.52 ± 0.05 | 0.46 ± 0.04* (150) |
| tMg (mmol/L) | 0.76 ± 0.08 | 0.48 ± 0.08* (150) | 0.77 ± 0.06 | 0.50 ± 0.06* (150) | 0.80 ± 0.06 | 0.60 ± 0.07* (130) | 0.78 ± 0.05 | 0.76 ± 0.06 (150) |
| Potassium (mmol/L) | 4.3 ± 0.3 | 3.4 ± 0.4* (150) | 4.2 ± 0.3 | 3.6 ± 0.2* (120) | 4.2 ± 0.3 | 3.6 ± 0.5* (120) | 4.6 ± 0.3 | 3.9 ± 0.3* (150) |
| Phosphate (mg/dL) | .4 ± 0.3 | 4.8 ± 0.7* (150) | 2.8 ± 0.3 | 4.1 ± 0.4* (150) | 2.8 ± 0.4 | 2.5 ± 0.4 (140) | 3.3 ± 0.3 | 3.5 ± 0.4 (120) |
| PTH (pmol/L) | 1.5 ± 0.5 | < 0.2* (20) | 1.0 ± 0.1 | < 0.2* (20) | 1.2 ± 1.2 | 1.3 ± 0.8 (120) | 0.9 ± 0.8 | 1.2 ± 0.7 (120) |
| Glucose (mg/dL) | 99 ± 109 | 3 ± 8 (120) | 100 ± 8 | 96 ± 12 (120) | 103 ± 7 | 164 ± 15* (120) | 106 ± 8 | 102 ± 10 (120) |
| Insulin (μU/mL) | 11.8 ± 6.0 | 15.0 ± 5.0 (120) | 10.3 ± 7.0 | 12.8 ± 6.2 (120) | 13.9 ± 3.5 | 52.0 ± 12.1* (90) | 11.2 ± 5.6 | 9.5 ± 4.8 (120) |
Infusions were administered for 120 minutes; initiation of infusion was designated as time 0 (baseline). Values in parentheses represent the number of minutes after initiation of an infusion at which the highest or lowest value was obtained.
Within a variable within an infusion solution, value differs significantly (P < 0.05) from the baseline value.
In horses infused with CaGlu (protocol 1), serum iCa and tCa concentrations increased in a linear manner from baseline to the end of the calcium infusion (Figure 1; Table 1). Hypercalcemia attributable to iCa was associated with a significant decrease in serum iMg concentrations, which reached lowest values at 140 minutes (20 minutes after maximal hypercalcemia; r = −0.84). Serum magnesium concentrations continued to decrease, even after serum calcium concentrations were returning toward baseline values. Serum tMg concentration also decreased significantly. Serum potassium concentration decreased significantly, and serum phosphate concentration increased significantly. Serum PTH concentrations decreased to extremely low or undetectable concentrations. We did not detect significant differences in blood pH and serum concentrations of bicarbonate, sodium, chloride, glucose, and insulin for protocol 1.
Mean ± SD serum concentrations of iCa (circles) and iMg (squares; A) and potassium (circles) and phosphate (squares; B) in 6 healthy horses with hypercalcemia experimentally induced by IV administration of a 23% CaGlu solution (protocol 1). The calcium solution was administered beginning at time 0 for 120 minutes (gray bar). Notice that hypercalcemia was associated with hypomagnesemia, hypokalemia, and hyperphosphatemia. *Within a variable, value differs significantly (P < 0.05) from the value for time 0.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD serum concentrations of iCa (circles) and iMg (squares; A) and potassium (circles) and phosphate (squares; B) in 6 healthy horses with hypercalcemia experimentally induced by IV administration of a 23% CaGlu solution (protocol 1). The calcium solution was administered beginning at time 0 for 120 minutes (gray bar). Notice that hypercalcemia was associated with hypomagnesemia, hypokalemia, and hyperphosphatemia. *Within a variable, value differs significantly (P < 0.05) from the value for time 0.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD serum concentrations of iCa (circles) and iMg (squares; A) and potassium (circles) and phosphate (squares; B) in 6 healthy horses with hypercalcemia experimentally induced by IV administration of a 23% CaGlu solution (protocol 1). The calcium solution was administered beginning at time 0 for 120 minutes (gray bar). Notice that hypercalcemia was associated with hypomagnesemia, hypokalemia, and hyperphosphatemia. *Within a variable, value differs significantly (P < 0.05) from the value for time 0.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
In horses infused with CaCl2 (protocol 2), hypercalcemia was associated with a significant decrease in serum iMg and tMg concentrations (r = −0.80; Figure 2; Table 1). Serum potassium concentrations decreased significantly, and serum phosphate concentrations increased significantly. Serum PTH concentrations decreased to extremely low or undetectable concentrations. We did not detect significant differences in blood pH and serum concentrations of bicarbonate, sodium, chloride, glucose, and insulin for protocol 2.
Mean ± SD serum concentrations of iCa (circles) and iMg (squares; A) and potassium (circles) and phosphate (squares; B) in 6 healthy horses with hypercalcemia experimentally induced by IV administration of a 10% CaCl2 solution (protocol 2). Notice that hypercalcemia was associated with hypomagnesemia, hypokalemia, and hyperphosphatemia. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD serum concentrations of iCa (circles) and iMg (squares; A) and potassium (circles) and phosphate (squares; B) in 6 healthy horses with hypercalcemia experimentally induced by IV administration of a 10% CaCl2 solution (protocol 2). Notice that hypercalcemia was associated with hypomagnesemia, hypokalemia, and hyperphosphatemia. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD serum concentrations of iCa (circles) and iMg (squares; A) and potassium (circles) and phosphate (squares; B) in 6 healthy horses with hypercalcemia experimentally induced by IV administration of a 10% CaCl2 solution (protocol 2). Notice that hypercalcemia was associated with hypomagnesemia, hypokalemia, and hyperphosphatemia. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
For protocols 1 and 2, as serum iCa concentrations increased, serum iMg concentrations decreased linearly (Figures 1–3). Even when serum iCa concentrations reached a peak at 120 minutes, serum iMg concentrations continued to decrease for another 20 minutes, which suggested that an Emax for magnesium was attained. Moreover, as long as serum iCa concentrations remained > 7.5 mg/dL, the return of iMg concentration to the reference range was slow; however, once serum iCa concentrations decreased to < 7.5 mg/dL, serum iMg concentrations returned to the reference range rapidly. Scatter plots of the mean serum iCa and iMg relationship after administration of both CaGlu and CaCl2 revealed hysteresis in which the values from induction of hypercalcemia were significantly higher than those from the recovery of hypercalcemia; this phenomenon was more evident for administration of CaGlu (protocol 1).
Scatter plots of the relationship between mean serum concentrations of iCa and iMg in healthy horses infused for 120 minutes with a 23% CaGlu solution (A) or 10% CaCl2 solution (B). Infusion of either calcium salt resulted in a linear relationship between iCa and iMg concentrations during the induction of hypercalcemia (closed circles; solid arrow), whereas the relationship was sigmoidal during recovery from hypercalcemia (open circles; dashed arrow). Notice that as long as serum iCa concentrations remained > 7.5 mg/dL, the return of iMg concentrations to baseline values was slow. Moreover, at similar iCa concentrations, iMg concentrations during the induction period were significantly higher than during the recovery period, a finding consistent with hysteresis. Notice that hysteresis was more evident for administration of CaGlu.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Scatter plots of the relationship between mean serum concentrations of iCa and iMg in healthy horses infused for 120 minutes with a 23% CaGlu solution (A) or 10% CaCl2 solution (B). Infusion of either calcium salt resulted in a linear relationship between iCa and iMg concentrations during the induction of hypercalcemia (closed circles; solid arrow), whereas the relationship was sigmoidal during recovery from hypercalcemia (open circles; dashed arrow). Notice that as long as serum iCa concentrations remained > 7.5 mg/dL, the return of iMg concentrations to baseline values was slow. Moreover, at similar iCa concentrations, iMg concentrations during the induction period were significantly higher than during the recovery period, a finding consistent with hysteresis. Notice that hysteresis was more evident for administration of CaGlu.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Scatter plots of the relationship between mean serum concentrations of iCa and iMg in healthy horses infused for 120 minutes with a 23% CaGlu solution (A) or 10% CaCl2 solution (B). Infusion of either calcium salt resulted in a linear relationship between iCa and iMg concentrations during the induction of hypercalcemia (closed circles; solid arrow), whereas the relationship was sigmoidal during recovery from hypercalcemia (open circles; dashed arrow). Notice that as long as serum iCa concentrations remained > 7.5 mg/dL, the return of iMg concentrations to baseline values was slow. Moreover, at similar iCa concentrations, iMg concentrations during the induction period were significantly higher than during the recovery period, a finding consistent with hysteresis. Notice that hysteresis was more evident for administration of CaGlu.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
In the induction phase of hypercalcemia, there was a negative linear relationship between serum iCa and iMg concentrations. The recovery phase was best fit by a sigmoidal relationship for Emax (Figure 3).25 This relationship also suggested that the serum iCa concentrations achieved in the study reported here were higher than the Emax that affected extracellular iMg concentrations.
In horses infused with dextrose solution (protocol 3), serum calcium concentrations did not change from baseline values. Serum magnesium and potassium concentrations decreased significantly (Table 1). Serum phosphate concentrations also decreased; however, this difference was not significant. Hyperglycemia was associated with a significant increase in insulin concentrations. There were no significant changes in blood pH and serum concentrations of bicarbonate, sodium, chloride, and PTH for protocol 3.
In horses infused with NaGlu (protocol 4), serum magnesium and potassium concentrations decreased significantly (Table 1). There was also a mild but significant decrease in serum calcium concentrations. We did not detect significant differences in blood pH and serum concentrations of bicarbonate, phosphate, sodium, chloride, PTH, glucose, and insulin for protocol 4.
In horses infused with saline solution, no significant changes were detected in serum concentrations of electrolytes, glucose, insulin, and PTH. Similarly, we did not detect significant differences in any urinary variables at any time point (data not shown).
The induction of hypercalcemia with CaGlu or CaCl2 resulted in a significant increase in FCa, FMg, FNa, FK, FCl, FPO4, and urine output, whereas urine osmolality and specific gravity decreased (Figures 4–6; Table 2). Urine pH decreased significantly in horses infused with CaCl2. For the duration of the infusion of CaGlu or CaCl2, there was an association between mean serum iCa concentrations and mean fractional excretion of electrolytes.
Mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A); FNa (circles), FK (squares), and FCl (triangles; B); and urine output (circles), osmolality (squares), and specific gravity (triangles; C) in 4 healthy horses with hypercalcemia experimentally induced by IV administration of a 23% CaGlu solution. Results were expressed as a percentage of the urinary excretion of creatinine. Notice that hypercalcemia increased FCa, FMg, FPO4, FNa, FK, and FCl; increased urine output; and decreased urine osmolality and specific gravity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A); FNa (circles), FK (squares), and FCl (triangles; B); and urine output (circles), osmolality (squares), and specific gravity (triangles; C) in 4 healthy horses with hypercalcemia experimentally induced by IV administration of a 23% CaGlu solution. Results were expressed as a percentage of the urinary excretion of creatinine. Notice that hypercalcemia increased FCa, FMg, FPO4, FNa, FK, and FCl; increased urine output; and decreased urine osmolality and specific gravity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A); FNa (circles), FK (squares), and FCl (triangles; B); and urine output (circles), osmolality (squares), and specific gravity (triangles; C) in 4 healthy horses with hypercalcemia experimentally induced by IV administration of a 23% CaGlu solution. Results were expressed as a percentage of the urinary excretion of creatinine. Notice that hypercalcemia increased FCa, FMg, FPO4, FNa, FK, and FCl; increased urine output; and decreased urine osmolality and specific gravity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A); FNa (circles), FK (squares), and FCl (triangles; B); and urine output (circles), osmolality (squares), and specific gravity (triangles; C) in 4 healthy horses with hypercalcemia experimentally induced by IV administration of a 10% CaCl2 solution. Notice that hypercalcemia increased FCa, FMg, FPO4, FNa, FK, and FCl; increased urine output; and decreased urine osmolality and specific gravity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A); FNa (circles), FK (squares), and FCl (triangles; B); and urine output (circles), osmolality (squares), and specific gravity (triangles; C) in 4 healthy horses with hypercalcemia experimentally induced by IV administration of a 10% CaCl2 solution. Notice that hypercalcemia increased FCa, FMg, FPO4, FNa, FK, and FCl; increased urine output; and decreased urine osmolality and specific gravity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A); FNa (circles), FK (squares), and FCl (triangles; B); and urine output (circles), osmolality (squares), and specific gravity (triangles; C) in 4 healthy horses with hypercalcemia experimentally induced by IV administration of a 10% CaCl2 solution. Notice that hypercalcemia increased FCa, FMg, FPO4, FNa, FK, and FCl; increased urine output; and decreased urine osmolality and specific gravity. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Relationship between mean ± SD serum iCa concentrations and mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A and C) and FNa (circles), FK (squares), and FCl (triangles; B and D) during induction of hypercalcemia in healthy horses achieved by IV administration of 23% CaGlu solution (A and B) and 10% CaCl2 solution (C and D) for 120 minutes. Notice that induction of hypercalcemia with CaGlu or CaCl2 was associated with an increase in FCa, FMg, FPO4, FNa, FK, and FCl. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Relationship between mean ± SD serum iCa concentrations and mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A and C) and FNa (circles), FK (squares), and FCl (triangles; B and D) during induction of hypercalcemia in healthy horses achieved by IV administration of 23% CaGlu solution (A and B) and 10% CaCl2 solution (C and D) for 120 minutes. Notice that induction of hypercalcemia with CaGlu or CaCl2 was associated with an increase in FCa, FMg, FPO4, FNa, FK, and FCl. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Relationship between mean ± SD serum iCa concentrations and mean ± SD FCa (circles), FMg (squares), and FPO4 (triangles; A and C) and FNa (circles), FK (squares), and FCl (triangles; B and D) during induction of hypercalcemia in healthy horses achieved by IV administration of 23% CaGlu solution (A and B) and 10% CaCl2 solution (C and D) for 120 minutes. Notice that induction of hypercalcemia with CaGlu or CaCl2 was associated with an increase in FCa, FMg, FPO4, FNa, FK, and FCl. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 5; 10.2460/ajvr.68.5.543
Mean ± SD urinary fractional excretion of electrolytes and other variables in horses infused with CaGlu, CaCl2, dextrose, and NaGlu solutions.
| Variable | CaGlu (protocol 1; n = 4) | CaCl2 (protocol 2; n = 6) | Dextrose (protocol 3; n = 3) | NaGlu (protocol 4; n = 3) | ||||
|---|---|---|---|---|---|---|---|---|
| Baseline | Infusion | Baseline | Infusion | Baseline | Infusion | Baseline | Infusion | |
| FCa (%) | 5.4 ± 2.5 | 56.2 ± 17.0* (120) | 5.4 ± 1.9 | 47.7 ± 10.8* (150) | .8 ± 2.2 | 5.1 ± 2.4 | 4.1 ± 1.2 | 10.7 ± 3.5* (150) |
| FMg (%) | 23.5 ± 5.8 | 55.0 ± 11.0* (120) | 28.5 ± 7.3 | 43.8 ± 9.0* (150) | 24.4 ± 6.5 | 12.5 ± 5.2* (180) | 24.3 ± 5.1 | 35.7 ± 7.8 (150) |
| FK (%) | 45.4 ± 18.5 | 116.6 ± 46.8* (150) | 38.4 ± 4.8 | 89.0 ± 14.0* (150) | .7 ± 12.4 | 30.1 ± 10.6 (150) | .6 ±11.2 | 65.0 ± 13.4 (150) |
| FPO4 (%) | 0.04 ± 0.04 | 0.50 ± 0.40* (120) | 0.14 ± 0.10 | 0.80 ± 0.50* (120) | 0.06 ± 0.12 | 0.04 ± 0.15 (150) | 0.05 ± 0.40 | 0.20 ± 0.40 (150) |
| FNa (%) | 0.09 ± 0.08 | 4.30 ± 2.00* (150) | 0.03 ± 0.01 | 4.80 ± 1.40* (120) | 0.05 ± 0.20 | 0.10 ± 0.20 (120) | 0.02 ± 0.03 | 1.20 ± 1.00* (120) |
| FCl (%) | 0.65 ± 0.27 | 5.60 ± 3.60* (120) | 0.70 ± 0.20 | 9.30 ± 2.70* (120) | 0.90 ± 0.08 | 1.20 ± 0.90 (120) | 0.60 ± 0.12 | 1.10 ± 0.10 (120) |
| Osmolality (m0sm/kg) | 1,240 ± 167 | 637 ± 170* (150) | 1,254 ± 94 | 618 ± 56* (150) | 1,275 ± 102 | 853 ± 97* (150) | 1,185 ± 150 | 1,090 ± 160 (150) |
| Output (mL/kg/h) | 1.10 ± 0.14 | 4.10 ± 0.70* (180) | 1.20 ± 0.35 | 3.20 ± 0.60* (150) | 1.30 ± 0.40 | 1.90 ± 0.50 (150) | 0.90 ± 0.40 | 1.30 ± 0.60 (150) |
| Specific gravity | 1.027 ± 0.005 | 1.007 ± 0.002* (150) | 1.029 ± 0.004 | 1.006 ± 0.002* (150) | 1.035 ± 0.005 | 1.020 ± 0.004* (120) | 1.032 ± 0.006 | 1.028 ± 0.007 (150) |
| pH | 8.1 ± 0.4 | 7.8 ± 0.4 (120) | 8.0 ± 0.2 | 7.3 ± 0.4* (120) | 8.1 ± 0.3 | 7.8 ± 0.4 (150) | 7.9 ± 0.3 | 7.6 ± 0.4 (120) |
See Table 1 for key
In horses infused with dextrose solution, we did not detect significant changes in FCa, FNa, FCl, FK, FPO4, and urine pH at any time point. The FMg decreased significantly (Table 2). The FK decreased but not significantly (P = 0.06). Urine osmolality and specific gravity also decreased significantly. Urine output increased; however, this difference was not significant (P = 0.06).
In horses infused with NaGlu, there was a significant increase in FCa and FNa (Table 2). We did not detect changes in urine output, osmolality, specific gravity, or urine pH in horses infused with NaGlu.
Discussion
In the study reported here, we found that the induction of hypercalcemia in healthy horses by the administration of CaGlu or CaCl2 resulted in hypomagnesemia, hypokalemia, and hyperphosphatemia; caused a decrease in PTH concentrations; caused an increase in FCa, FMg, FK, FPO4, FNa, and FCl; and induced diuresis. Infusions with NaGlu and dextrose solution decreased serum magnesium and potassium concentrations but to a lesser degree than the decreases caused by administration of calcium salts. No changes in serum glucose and insulin concentrations were detected in horses infused with CaGlu, CaCl2, or NaGlu. Only the infusion of dextrose solution increased serum glucose and insulin concentrations.
Several mechanisms should be considered to explain the development of hypomagnesemia during hypercalcemia in these horses. We speculate that the primary cause of hypomagnesemia was increased urinary wasting of magnesium as a result of decreased renal reabsorption of iMg. We propose that CaR activation in the TAL decreased the transepithelial transport of calcium, magnesium, potassium, sodium, and chloride by inhibiting the furosemide-sensitive Na+-K+-2Cl− cotransporter.1 Specifically, hypercalcemia decreased the voltage gradient required for paracellular reabsorption of calcium and magnesium and the transcellular reabsorption of sodium, potassium, and chloride. Because most magnesium resorption is in the TAL,3 which is influenced by PTH and CaR among other factors, it is reasonable to assume that changes in serum calcium concentrations can rapidly affect extracellular magnesium concentrations. Moreover, because both calcium and magnesium use similar albumin binding sites, and increases in serum calcium concentrations can displace magnesium from albumin,26 it is also possible that more magnesium can be ultrafiltered and wasted by the kidneys during hypercalcemia. In addition, the high calcium–low PTH concentrations likely decreased the transcellular transport of calcium and magnesium in the distal portion of the nephron by inhibiting the activity of epithelial cation channels.4
Hypomagnesemia could also have been the result of the gluconic acid in CaGlu inducing insulin release.2,27 However, to our knowledge, there is no information to suggest that gluconic acid induces insulin release, and we did not detect hyperglycemia or hyperinsulinemia in horses administered CaGlu or NaGlu. Of interest, intra-arterial infusion of CaGlu stimulates insulin release in humans with insulinomas but not in healthy people.28
Because gluconic acid has the ability to chelate calcium and magnesium, we also evaluated the effects of NaGlu on serum concentrations of magnesium and calcium. We found that an equivalent dose of NaGlu induced a mild decrease in serum concentrations of magnesium and potassium; however, its magnitude was less than the hypomagnesemia induced by administration of CaGlu or CaCl2. This finding is consistent with the low stability constant of gluconic acid determined by the National Institute of Standards and Technology17 for calcium and magnesium, compared with the stability constant for EDTA. Therefore, we hypothesize that the hypomagnesemia in horses administered CaGlu was primarily a calcium-induced process. However, we believe that gluconic acid played a role in the induction of hypomagnesemia in horses administered CaGlu because the hypomagnesemia induced with CaGlu was of greater magnitude than that induced by CaCl2.
Gluconic acid could also have contributed to the increase in FMg (probably as a result of chelation or low urinary pH) because there was a mild increase in FMg in horses administered NaGlu. Furthermore, it has been reported that a fraction of the gluconic acid administered parenterally as NaGlu to rats can be eliminated unchanged in urine.20,21
Another important factor to consider for the effects on serum and urinary electrolytes is the acid-base balance that results from changes in the cation-anion difference. We did not detect significant changes in blood pH in any of the experiments; however, urine pH in horses administered CaCl2 decreased. Investigators in another study29 reported that horses fed a diet with a low dietary cation-anion difference had a mild decrease in serum magnesium concentrations, lower urine pH, and decreased urinary excretion of magnesium, compared with results for horses fed a diet with a high dietary cation-anion difference; however, in dairy cows, feeding a diet with a low dietary cation-anion difference is associated with increased urinary excretion of calcium and magnesium.
Because insulin can induce hypomagnesemia, hypokalemia, and hypophosphatemia,2,3,27,30,31 we also evaluated the effects of glucose (and insulin release) on serum concentrations of magnesium, potassium, and phosphate concentrations. Infusion of a dextrose solution resulted in hyperglycemia, hyperinsulinemia, hypomagnesemia, and hypokalemia and a mild decrease in phosphate concentrations, which could have been the result of hyperinsulinemia.31 We did not detect significant changes in the urinary excretion of sodium, calcium, chloride, or phosphate; however, we detected a mild decrease in FK and FMg. Analysis of these findings indicates that the effects of dextrose (and insulin) are primarily mediated by intracellular shifts of magnesium and potassium and are not the result of urinary losses, as happens with calcium salts.
Similar to the effect for magnesium, we also believe that the effect of calcium to cause a decrease in serum concentrations of potassium is, in part, the result of increased urinary excretion of potassium, as previously discussed. However, we cannot rule out the possibility that hypercalcemia directly or indirectly caused an intracellular shift of potassium. In our experience, we have found that rapidly induced hypercalcemia (via administration of CaGlu or CaCl2) can cause a rapid decrease in serum potassium concentrations that cannot be explained by increased urinary excretion of potassium. However, in the study reported here, we did not detect changes in blood pH or bicarbonate concentrations for any of the protocols; therefore, hypokalemia was unlikely to be a result of metabolic alkalosis and subsequent intracellular shifts of potassium.
In the study reported here, hypercalcemia consistently resulted in hyperphosphatemia, which we believe was, in part, the result of urinary phosphate retention from a decrease in PTH concentrations because PTH is the main phosphaturic hormone.32 Mobilization of phosphate to the extracellular compartment to counteract high calcium concentrations could have contributed to hyperphosphatemia. Acute hypercalcemia in various species has been associated with increases in serum concentrations of phosphate that are independent of PTH,15,33,34 and the proposed mechanism is an intracellular efflux of phosphate.15,35 Hypercalcemia also increased the urinary excretion of phosphate, which was an unexpected finding because PTH concentrations were low. Analysis of data from other studies15,36 suggests that hypercalcemia increases the urinary excretion of phosphate by a PTH-independent mechanism, likely by decreasing the number of sodium-phosphate cotransporters in the proximal tubules. Phosphaturic hormones, such as calcitonin, which increase during hypercalcemia in many species (including, in our experience, horses), could have contributed to hyperphosphaturia.32,37
In addition to the aforementioned effects of CaR on electrolyte reabsorption in the TAL, hypercalcemia also inhibits Na+-K+ ATPase in the proximal tubules,38 which contributes to natriuresis and diuresis, as was evident in the study reported here. The diuretic properties of calcium are enhanced by CaR activation in the collecting ducts, where it reduces vasopressin-elicited water reabsorption.39 Hypercalcemia has natriuretic properties in humans and rats that can lead to hyponatremia and depletion of total body sodium and water.12,13,15
The study reported here also revealed the exquisite sensitivity of the calcium-sensing system in horses because an increase in serum calcium concentrations of approximately 10% from baseline values initiated a decrease in serum concentrations of magnesium and PTH and increased the urinary excretion of several electrolytes. These results resemble our findings for the calcium-PTH relationship in horses in which we determined that calcium concentrations > 7.2 mg/dL (1.8 mmol/L) had no additional inhibitory effects on PTH secretion.22 It is of interest that, similar to hysteresis for the calcium-PTH relationship in healthy horses,22 the calcium-magnesium relationship also resulted in hysteresis. Both processes are likely mediated by CaR. Although the induction segment of the calcium-magnesium relationship was linear, the recovery was better fit by a sigmoidal Emax relationship,25 which supports our belief that there was a CaR saturable phenomenon. Pharmacodynamic studies, including induction of various blood calcium concentrations, will be required to better understand the interactions of calcium with other electrolytes.
To our knowledge, the study reported here is the first in which the effect of hypercalcemia on the urinary excretion of electrolytes in horses has been evaluated and is one of the few studies to evaluate hypercalcemia, serum concentrations of magnesium, and urinary excretion of magnesium in any species.13 We reported that increasing serum calcium concentrations by 35% from baseline values increased the urinary excretion of electrolytes by several fold and induced diuresis. Results of the study also support the concept that sustained hypercalcemia can lead to depletion of electrolytes and volume depletion.12,13
Although there are limitations to our study, in particular the small sample size for the dextrose solution and NaGlu groups, we believe that not including these groups would have raised questions as to whether the observed effects on serum and urinary electrolytes were calcium-mediated events or resulted from hyperinsulinemia or gluconic acid chelation.
The findings reported here provide additional insight on the effects of extracellular calcium on several electrolytes. We often detect iCa values of > 10 mg/dL in horses with hypercalcemia attributable to malignancy, hyperparathyroidism, and renal failure. Humans and horses with disease-associated hypercalcemia can have polyuria and increased urinary wasting of electrolytes. Furthermore, both hypocalcemia and hypomagnesemia are frequent findings in horses with sepsis and gastrointestinal tract disease,10 and affected horses often receive solutions containing calcium salts and IV administration of fluids, both of which can increase renal tubular flow and urinary wasting of magnesium. The lack of food intake as a result of a disease process can worsen the magnesium status because magnesium is regulated, in part, by intestinal absorption. Despite the fact that hypomagnesemia is common in critically ill horses, treatment with magnesium salts is rarely practiced. Hypomagnesemia has been associated with an increase in fatalities in several species,11,40 including horses.7 Analysis of the results of the study reported here suggests that magnesium treatment should be considered for horses with certain pathologic processes. On the basis of the findings of our study, careful monitoring of serum concentrations of calcium and magnesium is warranted in humans and other animals administered calcium treatments and supports the use of calcium salts in emergency situations, such as severe hyperkalemia.
We believe the results of the study reported here have physiologic and clinical relevance. The study provided additional information to enhance our understanding of mammalian calcium and electrolyte interactions during health and disease.
ABBREVIATIONS
| iCa | Ionized calcium |
| PTH | Parathyroid hormone |
| CaR | Calcium-sensing receptor |
| iMg | Ionized magnesium |
| TAL | Thick ascending limb of the loop of Henle |
| CaGlu | Calcium gluconate |
| tCa | Total calcium |
| tMg | Total magnesium |
| FCa | Urinary fractional excretion of calcium |
| FMg | Urinary fractional excretion of magnesium |
| FNa | Urinary fractional excretion of sodium |
| FPO4 | Urinary fractional excretion of phosphate |
| FK | Urinary fractional excretion of potassium |
| FCl | Urinary fractional excretion of chloride |
| NaGlu | Sodium gluconate |
| Emax | Maximum calcium effect |
Vet/IV Infusion Pump, Heska Corp, Fort Collins, Colo.
Calcium gluconate 23% solution, Vedco Inc, St Joseph, Mo.
Calcium chloride, Sigma-Aldrich Corp, St Louis, Mo.
Dextrose 50% solution, Vedco Inc, St Joseph, Mo.
Sodium gluconate, Sigma-Aldrich Corp, St Louis, Mo.
Sodium chloride 0.9% solution, Baxter Healthcare Corp, Deerfield, Ill.
BD Angiocath, Becton-Dickinson, Sandy, Utah.
Cell-Dyn 3500, Abbott Diagnostics, Santa Clara, Calif.
Boehringer Mannheim/Hitachi 911 system, Boehringer Mannheim Corp, Indianapolis, Ind.
Vitros DT60 II, Ortho-Clinical Diagnostics, Rochester, NY.
Nova 8, Nova Biomedical, Waltham, Mass.
Immulite PTH, Diagnostic Products Corp, Los Angeles, Calif.
Coat-A-Count insulin, Diagnostic Products Corp, Los Angeles, Calif.
ACL 200 automated coagulation laboratory, Instrumentation Laboratory, Lexington, Mass.
ABL 500, Radiometer Medical A/S, Copenhagen, Denmark.
SigmaStat, version 3.0, SPSS Inc, Chicago, Ill.
JMP, version 5.1, SAS Institute Inc, Cary, NC.
SigmaPlot, version 9.0, SPSS Inc, Chicago, Ill.
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