Introduction

Intravenous magnesium sulfate (MgSO₄) is used in equine medicine to treat dysrhythmias, neurological disorders, hypomagnesemia in critical illness, and refractory hypocalcemia [1]. More recently, intravenous MgSO₄ has been used in equine events as a calming and performance-enhancing agent, with the ultimate goal of giving a competitive edge to horses that receive it [2,3].

Magnesium (Mg) is an essential macroelement involved in a multitude of physiological processes, including enzymatic activation, intermediary metabolism of carbohydrates, fats, and proteins, nucleic acid synthesis, cell membrane ion transport, neuromuscular excitability, cell proliferation, and calcium homeostasis [1]. The secretion of PTH and activity of the PTH receptor are modulated by Mg. Thus, Mg depletion could impair calcium regulation [4].

Similar to total calcium (Ca), in which ionized calcium (Ca²⁺) represents the active fraction, ionized Mg (Mg²⁺) is the active fraction of total Mg. Considering that a multitude of cellular processes are highly dependent on Mg, intracellular Mg concentrations are tightly regulated [1]. However, extracellular Mg²⁺ is not under tight hormonal homeostatic control as is Ca²⁺, and its plasma concentrations depend on gastrointestinal absorption, renal excretion, and bone exchange [1,5]. A number of hormonal and non-hormonal factors influence extracellular Mg²⁺ concentrations [6].

Parathyroid hormone (PTH) and calcitonin (CT) are important Ca-regulating hormones [7,8]. Hypocalcemia stimulates and hypercalcemia suppresses PTH secretion, while the opposite occurs with CT release [9]. In addition to its effects on Ca²⁺ homeostasis, PTH stimulates renal reabsorption of Mg²⁺ and its release from bone [10]. Calcitonin (CT) is secreted by the thyroid gland C cells in response to hypercalcemia to restore normocalcemia by blocking osteoclast-mediated bone resorption and reducing renal calcium reabsorption. There is evidence in other species that hypermagnesemia also stimulates CT secretion [11,12].

The ability of the parathyroid gland chief cells and thyroid gland C cells to detect extracellular Ca²⁺ concentrations depends on the calcium-sensing receptor (CaSR), a G-protein-coupled cell surface receptor [13,14]. The CaSR is also expressed in the renal tubular cells where it regulates the reabsorption of Ca²⁺, Mg²⁺, Na⁺, K⁺, Cl^-, and water in various species, including the horse [4,13,15]. Excessive CaSR activation promotes diuresis and could lead to dehydration in patients with chronic hypercalcemia [4,15]. Relevant to this study, the CaSR also detects other polyvalent cations, including Mg²⁺, Sr²⁺, Gd³⁺, and neomycin [16]. Therefore, hypermagnesemia could potentially influence PTH and CT secretion, plasma Ca²⁺ concentrations, as well as the urinary excretion of electrolytes.

Hypercalcemia affects calcium-regulating hormones, as well as plasma and urinary electrolytes in horses [4]. In addition, hypercalcemia decreases Mg²⁺ concentrations in horses [4]. Therefore, based on the Ca²⁺ and Mg²⁺ interactions, as well as the ability of Mg²⁺ to activate CaSR, one can speculate that hypermagnesemia could affect a number of blood and urinary variables in horses, which could have clinical implications, including calcium homeostasis, urinary electrolyte wasting, neuromuscular excitability, and cardiovascular function.

The goal of this study was to investigate the effect of experimentally induced hypermagnesemia with intravenous MgSO₄ on blood PTH, CT, Mg²⁺, Ca²⁺, Na⁺, K⁺, and Cl^- concentrations, as well as on their urinary excretion in healthy horses. We hypothesized that hypermagnesemia will reduce Ca²⁺ concentrations, initially suppress to later stimulate PTH secretion, with the opposite effects on CT concentrations. We also proposed that hypermagnesemia, similar to hypercalcemia, will increase the urinary fractional excretion of Mg²⁺, Ca²⁺, Na⁺, K⁺ and Cl^- in horses.

Materials and methods

Results

Plasma Mg²⁺, Ca²⁺, Na⁺, K⁺, and Cl^- concentrations and Mg²⁺/ Ca²⁺

Plasma Mg²⁺ concentrations increased sharply by 5 minutes and remained significantly higher than baseline for 180 minutes (Fig 1A and Table 1; P < 0.05). At 5, 15 and 60 minutes, plasma Mg²⁺ concentrations were 3.7, 2.8, and 1.7-fold baseline values, respectively (P < 0.05). At 360 minutes, 4 horses still had Mg²⁺ concentrations that were 30% higher than their baseline values. Hypermagnesemia led to a sharp and short lasting increase in plasma Ca²⁺ concentrations to then cause a steady and statistically significant decrease in Ca²⁺ concentrations (P < 0.05), reaching its lowest values at 45 minutes (12.5% decrease from time 0) to subsequently return to baseline values at 120 minutes (Fig 1A and Table 1). Plasma Na⁺, K⁺, Cl^-, and creatinine concentrations and plasma osmolality did not change over time (Table 1). The Mg²⁺/Ca²⁺ ratio was elevated by 5 minutes and remained above baseline values for 120 minutes (Table 1; P < 0.05).

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Fig 1.

Plasma Mg²⁺ and Ca²⁺ (A) and serum PTH and CT (B) concentrations following IV administration of MgSO₄ (60 mg/kg) in healthy horses. Values expressed as mean with S.E. * indicates statistically different than time 0. Rectangle over X axis represents IV administration of MgSO₄ over 5 minutes. Mg²⁺, plasma ionized magnesium; Ca^2+, plasma ionized calcium PTH, parathyroid hormone; CT, calcitonin.

https://doi.org/10.1371/journal.pone.0247542.g001

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Table 1. Plasma Mg²⁺, Ca²⁺, Na⁺, K⁺ and Cl^-, and serum PTH and CT concentrations, and Mg⁺²/Ca²⁺ ratio following IV administration of MgSO₄ (60 mg/kg) in healthy horses.

https://doi.org/10.1371/journal.pone.0247542.t001

Urinary electrolytes and osmolality

Urine Mg²⁺ concentrations increased steadily to reach statistical significance at 120 and 180 minutes compared to time 0 (Table 2; P < 0.05), to decrease to baseline values by 240 minutes. Urinary Ca²⁺ concentrations also increased by 180 minutes (Table 2; P < 0.05), to return to baseline at 240 minutes. Urine Na⁺ concentrations increased at 60 minutes, while urine K⁺ concentrations decreased at 30 minutes and returned to baseline at 360 minutes (Table 2; P < 0.05). Urinary Cl^- concentrations did not change overtime. Urinary osmolality decreased at 30 and 60 minutes to return to baseline thereafter (Table 2; P < 0.05).

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Table 2. Urine Mg²⁺, Ca²⁺, Na⁺, K⁺, Cl^-, and creatinine concentrations and urine osmolality following IV administration of MgSO₄ (60 mg/kg) in healthy horses.

https://doi.org/10.1371/journal.pone.0247542.t002

Urinary fractional excretion of electrolytes

The FMg increased by 3-fold at 30–120 minutes after MgSO₄ administration, then returned to baseline values by 360 minutes (Fig 2A; Table 3; P < 0.05). The FCa, FNa, FK, and FCl increased statistically by 60 minutes, and returned to baseline thereafter (Fig 2A and 2B; Table 3; P < 0.05).

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Fig 2.

FMg and FCa (A) and FNa, FK and FCl (B) (%) following IV administration of MgSO₄ (60 mg/kg) in healthy horses. Values expressed as mean with S.E. * indicates statistically different than time 0 (P < 0.05). Rectangle over X axis represents IV administration of MgSO₄ over 5 minutes. FMg, urinary fractional excretion of Mg²⁺; FCa, urinary fractional excretion of Ca²⁺; FNa, urinary fractional excretion of Na⁺; FK, urinary fractional excretion of K⁺; FCl, urinary fractional excretion of Cl^-.

https://doi.org/10.1371/journal.pone.0247542.g002

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Table 3. Urinary fractional excretion of Mg²⁺, Ca²⁺, Na⁺, K⁺, and Cl^- following IV administration of MgSO₄ (60 mg/kg, IV) in healthy horses.

https://doi.org/10.1371/journal.pone.0247542.t003

Discussion

In the present study, experimentally-induced hypermagnesemia in healthy horses led to a short lasting decrease in Ca²⁺ concentrations, with transient changes in PTH and CT concentrations, increasing the urinary fractional excretion of Mg²⁺, Ca²⁺, Na⁺, K⁺, and Cl^-, and decreasing urine osmolality.

A single intravenous dose of MgSO₄ at 60 mg/kg resulted in sustained hypermagnesemia for over 120 minutes. Similar results were observed in horses in which MgSO₄ was evaluated as a treatment for trigeminal headshaking syndrome and in human patients with preeclampsia [18,19]. These findings also suggest that the in horses the physiological effects of the single dose of MgSO₄ used in this study could last up to 6 hours. In addition, this information may be relevant to venues such as equestrian competitions, knowing that for at least 60 minutes after administration of this dose, serum [Mg²⁺] will be twice baseline values, potentially explaining the calming effects of hypermagnesemia in horses [2,3], which has potential regulatory connotations. An increase in Mg²⁺ concentrations coupled with a high Mg²⁺/Ca²⁺ ratio could be a potential biomarker for nefarious administration of MgSO₄ to competing horses [3,20]. It was also demonstrated that hypermagnesemia reduced headshaking behavior in horses [19]. These actions of Mg have been attributed to its ability to block N-methyl-D-aspartate (NMDA) ionotropic receptors and voltage-dependent calcium channels, reducing neuronal excitability [1,2,21,22]. The inhibitory effect of Mg²⁺ over NMDA receptor activity has also been the rationale for its use in acute brain injury, nociception, anesthesia, and behavioral disorders [1–3,19,21].

A decrease in Ca²⁺ concentrations after MgSO₄ infusion has been documented in human patients, horses and dogs [3,19,23–26] and could be attributed to excessive Mg²⁺ activation of CaSR in the renal tubules, increasing the urinary excretion of Ca and Mg, as reported in human patients [23,26,27]. We anticipated this response based on a previous study where experimental hypercalcemia decreased Mg²⁺ in healthy horses [4]. It is also possible that hypermagnesemia suppressed PTH secretion by activating CaSR [23], at least at early time points in the horses of this study, because subsequently there was a PTH rebound response that we attributed to the drop in Ca²⁺ concentrations. One difference between this study and publications using MgSO₄ as a tocolytic agent was the rapid intravenous administration of MgSO₄ to these horses compared to how it is administered to pregnant women [28]. One study in healthy horses showed that hypercalcemia has a similar effect as hypermagnesemia, increasing the urinary excretion of Ca²⁺, Mg²⁺, Na⁺, K⁺, and Cl^-, increasing urine output, and decreasing urine osmolality [4].

Parathyroid hormone promotes renal calcium reabsorption and bone resorption to maintain normocalcemia [29,30]. The PTH receptor, via cAMP, mediates the renal actions of PTH by increasing the activity of the Na⁺/K⁺/2Cl^- type 2 cotransporter (NKCC2), raising the electropositivity of the renal tubular lumen to promote paracellular cation reabsorption [31]. Activation of CaSR interferes with NKCC2, similar to furosemide, and inhibits vasopressin-mediated traffic of aquaporin-containing vesicles, explaining the diuretic actions of hypercalcemia in multiple species [15,32,33]. Under a similar premise supported by physiological studies showing that Mg²⁺ activates CaSR [13], hypermagnesemia could interfere with PTH secretion and renal reabsorption of electrolytes and water. In fact, CaSR has been described as a promiscuous receptor because it can sense multiple cations at different extracellular concentrations [13].

The initial PTH suppression could be attributed to excessive CaSR activation by Mg²⁺. This effect was likely counteracted by the drop in Ca²⁺ concentrations, which likely triggered a rebound PTH response, reflecting the preference of CaSR for Ca²⁺ over Mg²⁺ [13]. The increase in serum PTH concentrations occurred in parallel with the decrease in Ca²⁺ concentrations, supporting the rapid response of this system to protect against hypocalcemia [34,35]. After its rapid rise, CT concentrations returned to baseline values for the rest of the study, despite hypermagnesemia. This indicates that Mg²⁺ stimulated the secretion of CT stored in granules but was not sufficient to stimulate additional synthesis and secretion, in particular in the presence of low to normal Ca²⁺ concentrations. It is likely that a mechanism similar to that which occurred in the parathyroid gland chief cells, but with opposite endocrine secretion, was involved in thyroid gland C cell CT secretion. A similar CT response was observed in pigs after magnesium chloride infusion [36].

In addition to the increased FMg and FCa, the most feasible explanation for increased FNa, FK, and FCl were the effects of increased extracellular Mg²⁺ on transepithelial ion transport and water reabsorption, as previously discussed [37,38].

While no effect of hypermagnesemia on plasma Na⁺, K⁺, and Cl^- concentrations was documented, its evident effect on urinary electrolyte excretion indicates that in the short term, other homeostatic systems could maintain their concentrations. Evaluating the effect of prolonged hypermagnesemia on calcium-regulating hormones and electrolyte balance could provide additional insight on equine Mg²⁺ physiology.

The clinical implications of short-term hypermagnesemia on behavior, cardiovascular function, and neuromuscular activity in horses were recently documented [2,3,19], further demonstrating that Mg is a pleiotropic ion. Magnesium supplementation is recommended for a number of human disorders and is being promoted as a pre-anesthetic to reduce pain [21]. In addition, due to its calcium-channel and NMDA receptor blocking properties and prolonged half-life in horses, MgSO₄ supplementation should be considered for conditions associated with nociception, neuronal hyperexcitability, and energy dysregulation [1,2,21]. However, it is important to emphasize that intravenous administration of MgSO₄ to horses to give a competitive edge is a nefarious and dangerous practice.

The effect of intravenous MgSO₄ at the dose used in this study has not been prospectively evaluated in hospitalized horses, although lower doses of MgSO₄ are routinely used in equine patients with hypomagnesemia or dysrhythmias. It is anticipated that similar effects to the horses of this study would be expected in these patients. Oral magnesium supplements are used by horse owners and trainers to enhance systemic health and in general considered a low risk of causing hypermagnesemia because doses are low. One can speculate that chronic hypermagnesemia in horses could alter calcium and electrolyte homeostasis as well as neuromuscular activity, however, high Mg doses for a long time would be required. This would be a valid question to investigate to further understand Mg biology in healthy and sick horses.

Conclusions

A single intravenous bolus administration of MgSO₄ to healthy horses causes prolonged hypermagnesemia, a transient decrease in Ca²⁺ concentrations, and rapid changes in calcium-regulating hormones, increasing the urinary excretion of electrolytes and decreasing urine osmolality. This information enhances our understanding of equine Mg and Ca biology and has clinical implications. For example, based on information from this study, it would be of interest to know whether prolonged hypermagnesemia from excessive Mg supplementation leads to electrolyte depletion, water wasting, altered calcium homeostasis, with subsequent clinical consequences, including changes in neuromuscular activity, cardiovascular function, and behavior.