Plasma magnesium

Despite the absence of a (known) specific regulatory system, Mg²⁺ in plasma is kept within the range of 0·9–1·2 mmol/l, provided that influx (via absorption) into the extracellular space (ECS) including plasma is larger than the efflux (requirement and excretion). In humans, six genomic regions have been implicated in the maintenance of plasma Mg²⁺ concentration⁽ Reference Meyer, Verwoert and Hwang ^{15 )}, and similar gene loci may explain the heritability of plasma Mg²⁺ concentration in dairy cows (0·20–0·43)⁽ Reference Tsiamadis, Banos and Panousis ^{16 )}.

Plasma Mg²⁺ is known to be influenced in a non-specific manner by catecholamines⁽ Reference Rayssiguier ^{17 )}, insulin⁽ Reference Persson and Luthman ^{18 )} and parathyroid hormone (PTH)⁽ Reference Goff, Littledike and Horst ^{19 )}. In addition, epidermal growth factor has recently been shown to have magnesiotropic effects via TRPM6 channels, which regulate renal and intestinal Mg²⁺ absorption⁽ Reference Groenestege, Thebault and van der Wijst ^{20 )}.

Distribution of magnesium

Some 60–70 % of total body Mg²⁺ is bound in the skeleton. A further 30 % is found in the intracellular space (ICS) but only 1–5 % of intracellular Mg²⁺ is in the ionised form⁽ Reference Houillier ^{21 )}. The Mg²⁺ in the ECS only reflects about 1 % of total body Mg²⁺. Between 20 and 40 % of plasma Mg²⁺ is bound to albumin and globulin and some 10 % complexes with small anions such as citrate, phosphate and bicarbonate, so that 50–70 % are ionised.

According to a classical estimate⁽ Reference Blaxter and McGill ^{22 )}, the total content of Mg²⁺ within the body of calves can be calculated from:

(1) $${\rm Mg}\,\left( {\rm g} \right)\, {\equals}\, \left( {0\!\cdot\!{\rm 655}{\times}{\rm BW\ (kg)}} \right)-{\rm 3}\!\cdot\!{\rm 5},$$

where BW is body weight in kg.

Provided that a similar relationship holds for adult ruminants, the total body Mg²⁺ of a cow with a BW of 700 kg should be roughly 455 g, of which approximately 320 g would be skeletal, about 130 g intracellular, while only about 4–5 g would be found in the total ECS.

Regulation of magnesium homeostasis

The range of plasma Mg²⁺ primarily depends on the influx of Mg²⁺ from the gastrointestinal tract into the ECS (a) and on the efflux from the ECS into milk (b), into the ICS including soft tissue and bones during growth and the fetus during pregnancy (c), and into the intestine as endogenous secretion (d). Mg²⁺ not required for b–d is excreted into urine (e). Plasma Mg²⁺ concentration thus depends on the daily Mg²⁺ balance and is given by:

(2) $${\rm a\, {\equals}\, b{\plus}c{\plus}d{\plus}e,}$$

where a is Mg²⁺ absorption (g/d) (influx), b is Mg²⁺ efflux in milk (g/d), c is Mg²⁺ uptake (efflux) into the ICS (g/d), d is intestinal Mg²⁺ secretion (efflux) (g/d), and e is renal Mg²⁺ excretion (efflux) (g/d).

A scheme of Mg²⁺ metabolism for a cow with a BW of 700 kg and a milk production of 40 kg/d is given in Fig. 1.

Fig. 1 Scheme of Mg²⁺ metabolism in a non-pregnant dairy cow of 700 kg body weight (BW). The daily Mg²⁺ intake is 50 g, with true Mg²⁺ absorption being 12·8 g/d (25·6 %). The true absorption is reduced by an endogenous (Endog.) secretion of 2·8 g/d (4 mg/kg), which accounts for an apparent absorption or Mg²⁺ digestion of 10 g/d (20 %). An amount of 14·8 g Mg²⁺/d is used for 40 kg milk secretion (120 mg/l) and the surplus (5·2 g/d) is excreted via the kidneys into urine. The pool in the extracellular space has been calculated by assuming that the plasma volume and interstitial space represent 5 and 15 % of BW, respectively, as in sheep⁽ Reference Storry ^{26 )}. The unidirectional flow of Mg²⁺ into and out of the intracellular space (ICS) and bone is not known and net flux into the ICS or bone is zero at constant BW. In pregnant cows in late gestation a flux of 0·2 g Mg²⁺/d towards the fetus has to be included⁽ Reference House and Bell ^{207 )}.

In an adult and not growing, non-pregnant cow, net uptake of Mg²⁺ into the ICS and bone at adequate Mg intake is marginal, so that equation (2) can be simplified to:

(3) $${\rm a}\, {\equals}\, {\rm b}{\plus}{\rm d}{\plus}{\rm e},$$

where a is Mg²⁺ absorption (g/d) (influx), b is Mg²⁺ efflux in milk (g/d), d is endogenous Mg²⁺ secretion (efflux) (g/d), and e is renal Mg²⁺ excretion (efflux) (g/d).

Because Mg²⁺ absorption (a) rarely equals Mg²⁺ efflux, additional mechanisms are necessary for the adjustment, which is very efficiently controlled by the kidneys at Mg²⁺ surplus. However, mobilisation from the skeleton or the cytosol is very limited⁽ Reference Houillier ^{21 )}. This suggests that Mg²⁺ influx was very rarely limited during evolution. Obviously, Mg²⁺ intake and absorption (a) were generally above requirement (b + c + d), so that an efficient renal excretion of the surplus was sufficient for the regulation of Mg²⁺ homeostasis (e). Moreover, Mg²⁺ is not very toxic, and hence transient hypermagnesaemia (rapid influx>efflux) is well tolerated⁽ Reference Martens and Stössel ^{23 )}. Therefore, absorption from the gastrointestinal tract is the key factor determining plasma Mg²⁺ levels, which can only be kept constant when the daily requirement is replaced by an adequate absorption. A better comprehension of the gastrointestinal absorption (influx) and its large variation⁽ Reference Schonewille, Everts and Jittakhot ^{24 )} appears to be a key factor for understanding Mg²⁺ homeostasis.

Site of magnesium absorption

Early studies in vivo suggested the distal part of the small intestine as the site of Mg²⁺ absorption⁽ Reference Smith ^{25 )} and Storry⁽ Reference Storry ^{26 )} stated that ‘there is no evidence to assume that ... a mechanism exists for the transport of calcium and magnesium across the rumen epithelium’. In contrast, Harrison et al. ⁽ Reference Harrison, Hill and Mangan ^{27 )} observed Mg²⁺ absorption proximal to the duodenum and Marongiu from the rumen fluid⁽ Reference Marongiu ^{28 )}. In a later study, Rogers & van’t Klooster⁽ Reference Rogers and van’t Klooster ^{29 )} measured mineral flow rates along the gut and demonstrated absorption before the duodenum. Martens⁽ Reference Martens ^{30 )} summarised data from sheep and cows. The rate of absorption before the duodenum increased linearly with Mg²⁺ intake⁽ Reference Martens ^{31 )}. A net secretion was observed into the small intestine; the same amount was absorbed in the large intestine.

Regarding the location of Mg²⁺ absorption before the duodenum, uptake from the omasum but not from the abomasum was demonstrated⁽ Reference Edrise and Smith ^{32 ,} Reference Field and Munro ^{33 )}. However, Martens & Rayssiguier⁽ Reference Martens and Rayssiguier ^{34 )} showed that, in vitro, the transport capacity of the rumen epithelium was large and predominant.

Mechanism of ruminal Mg²⁺ transport

Scott⁽ Reference Scott ^{40 )} analysed the passive driving forces across the rumen epithelium and concluded that the chemical gradient of Mg²⁺ for passive movement from the rumen to plasma was opposed by the stronger electrical gradient (blood side positive 30–60 mV), which thus prevented passive paracellular uptake of the Mg²⁺ ion from the rumen to blood. Accordingly, Mg²⁺ transport across the rumen epithelium has to be energised.

The exclusion of passive paracellular diffusion suggests active, transcellular transport, which was deduced both from in vitro and in vivo experiments⁽ Reference Martens and Harmeyer ^{41 –} Reference Martens ^{43 )} showing: (a) net transport of Mg²⁺ from the rumen to blood; (b) saturation kinetics; (c) significantly lower transport at lower temperature; and (d) reduced transport by inhibition of Na⁺/K⁺-ATPase (ouabain or dinitrophenol). Furthermore, it was shown that ‘bulk flow’ could not explain ruminal Mg²⁺ transport⁽ Reference Martens ^{44 )}, as had been proposed for rats⁽ Reference Behar ^{45 )}.

The movement of Mg²⁺ across the multilayered epithelium includes: (a) uptake across the apical membrane; (b) the passage of Mg²⁺ across the various epithelial layers of the multilayered epithelium; and (c) release across the basolateral membrane. In cases where the passive gradients are sufficient, transepithelial transfer may further involve (d) possible paracellular passive movement.

Epithelial mechanisms

For many decades, characterisation of the transcellular transport pathway suffered from a lack of knowledge about Mg²⁺ transport. The existence of Mg²⁺ ion channels was still widely considered an unproven hypothesis. However, transport mechanisms for other ions (for example, Na⁺ or Ca²⁺) had been clearly established in other epithelia, such as in rabbit ileum⁽ Reference Frizzell and Schultz ^{46 )}. Simply put, transport of ions across epithelia is either influenced by the transepithelial potential difference (PD_t) or not. By varying the electrical driving force for a specific ion (ξ)⁽ Reference Frizzell and Schultz ^{46 )}, and plotting the measured flux rate over ξ, it is possible to differentiate between the PD-independent flux (given by the y-intercept in the plot) and the PD-dependent flux (given by the slope of the plotted curve).

Potential-dependent Mg²⁺ uptake

The suggestion that Mg²⁺ transport occurred with the passage of a charge was deduced from a reciprocal relationship between an increase in ruminal PD_t and a decrease in ruminal Mg²⁺ transport⁽ Reference Tomas and Potter ^{36 ,} Reference Martens, Gäbel and Strozyk ^{47 )}. PD_t can be calculated from the apical potential difference (PD_a) and the basolateral potential difference (PD_b): PD_t=PD_a – PD_b. In this relationship, the sign convention is such that the apical (ruminal) side is set to ground level and an increase in the passage of cations from the apical to the serosal (blood) side will lead to a more positive PD_t and a less negative PD_a, thus reducing the driving force for apical Mg²⁺ uptake. Mucosal to serosal Mg²⁺ transport rates (J_ms Mg²⁺) revealed a linear correlation between ξ (PD_t) and J_ms Mg²⁺ within –25 and + 25 mV⁽ Reference Leonhard-Marek and Martens ^{48 )}. The obtained slope confirmed the suggestion of PD-dependent J_ms Mg²⁺ transport with uptake as an ion (for example, channel-mediated), but also exhibited an intercept of the y-axis, which represents a PD-independent component (for example, via co-transport).

A PD-dependent uptake mechanism for Mg²⁺ in the apical membrane is supported by data from microelectrode experiments. Leonhard-Marek & Martens⁽ Reference Leonhard-Marek and Martens ^{48 )} measured a PD_a under open circuit conditions of –67·3 mV (cytosol negative). An increase in the mucosal K⁺ concentration depolarised PD_a and increased PD_t. These experiments suggested that the apical membrane is permeable to K⁺, with non-selective cation channels from the TRP family such as TRPV3 and TRPA1 likely candidates⁽ Reference Rosendahl, Braun and Schrapers ^{10 )}.

In further flux measurements, Mg²⁺ transport was reduced not only by elevation of the PD_t but also by the apical K⁺ concentration⁽ Reference Martens, Gäbel and Strozyk ^{47 ,} Reference Leonhard-Marek and Martens ^{48 )}. Depolarisation of PD_a by K⁺ is the most likely explanation for the reduced mucosal to serosal flux of Mg²⁺ (J_ms) at high concentrations of K⁺ (80 mmol/l) and argues for the uptake of Mg²⁺ by a PD-dependent mechanism. Since the ionised intracellular Mg²⁺ concentration (0·54 mmol/l)⁽ Reference Schweigel, Lang and Martens ^{49 )} is lower than the concentration of Mg²⁺ in the rumen, the uptake of Mg²⁺ is driven by the electrochemical gradient across the apical membrane.

The ruminal Mg²⁺ channel

The significant correlation between changes of PD_a and Mg²⁺ transport led to the suggestion of an apical Mg²⁺ channel⁽ Reference Martens, Gäbel and Strozyk ^{47 )}, long before such channels were cloned. Channel-mediated transport of Mg²⁺ is now well established⁽ Reference Hoenderop and Bindels ^{50 )}. Thus, hypomagnesaemia in man is now known to be caused by the mutation of a channel of the TRP gene family, TRPM6⁽ Reference Konrad, Schlingmann and Gudermann ^{51 )}. TRPM6 plays a key role in the intestinal and renal absorption of Mg²⁺ in mice⁽ Reference Hoenderop and Bindels ^{50 )}. Expression of mRNA encoding for this protein by the rumen epithelium suggests a similar role in the ruminal absorption of Mg^{2+ (} Reference Rosendahl, Braun and Schrapers ^{10 )}.

A further member of this channel family, TRPM7, has been demonstrated in ruminal epithelial cells as mRNA⁽ Reference Rosendahl, Braun and Schrapers ^{10 ,} Reference Schweigel, Kolisek and Nikolic ^{52 )} and protein⁽ Reference Schweigel, Kolisek and Nikolic ^{52 )} and is thought to play a role in intracellular Mg²⁺ homeostasis⁽ Reference Demeuse, Penner and Fleig ^{53 )}. Since experiments in TRPM7-deficient mice by Ryazanova et al. ⁽ Reference Ryazanova, Rondon and Zierler ^{54 )} demonstrate disturbed intestinal Mg²⁺ absorption, an additional role in epithelial transport has been proposed. It has been suggested that both candidate genes are of functional importance for epithelial transport since both TRPM6 and TRPM7 subunits may be required to form a functional Mg²⁺ channel⁽ Reference Dimke, Hoenderop and Bindels ^{55 )}. MagT1 is a further candidate gene for the PD-dependent uptake pathway in ruminal epithelial cells⁽ Reference Schweigel, Kolisek and Nikolic ^{52 ,} Reference Schweigel, Kuzinski and Deiner ^{56 )}.

Potential difference-independent (electroneutral) Mg²⁺ uptake

In addition to the channel-mediated pathway, a second, PD-independent Mg²⁺ uptake pathway mediates Mg²⁺ transport⁽ Reference Leonhard-Marek and Martens ^{48 )}. The charge of Mg²⁺ is compensated by co-transport with anions or counter-transport of cations. Interestingly, the intake of high levels of readily fermentable carbohydrates⁽ Reference Giduck and Fontenot ^{57 )} increased Mg²⁺ digestion. Furthermore, SCFA or CO₂ enhanced ruminal Mg²⁺ absorption in vivo ⁽ Reference Martens, Heggemann and Regier ^{58 )} and stimulated J_ms Mg²⁺ in vitro ⁽ Reference Leonhard-Marek, Gäbel and Martens ^{59 )}. Since both fermentation products acidify the epithelium, Mg²⁺/2H⁺ exchange has been proposed to represent this transport mechanism⁽ Reference Leonhard-Marek, Gäbel and Martens ^{59 ,} Reference Leonhard-Marek ^{60 )}.

However, Schweigel & Martens⁽ Reference Schweigel and Martens ^{61 )} found no experimental evidence for directly coupled Mg²⁺/2H⁺ exchange in isolated ruminal epithelial cells of sheep and suggested a co-transport of Mg²⁺ with an anion such as HCO₃ ^– or Cl^–. Furthermore, the conductance of ruminal TRP channels for monovalent cations is activated by exposure to SCFA, possibly related to swelling of the cells⁽ Reference Stumpff, Martens and Bilk ^{62 ,} Reference Georgi, Rosendahl and Ernst ^{63 )}. This opens the possibility that the stimulation of Mg²⁺ by SCFA and CO₂ may not exclusively represent PD-independent Mg²⁺ transport but also involves stimulation of PD-dependent mechanisms. Finally, the activity of the ruminal vacuolar H⁺-ATPase modulates Mg²⁺ transport⁽ Reference Schweigel and Martens ^{61 )}, possibly by increasing PD_a and thus enhancing the uptake of Mg²⁺. Such a mechanism would represent functional albeit not fixed Mg²⁺/H⁺ exchange. Currently, neither the stoichiometry nor the molecular identity of the PD-independent Mg²⁺ transporter is known.

Physiological consequences of two uptake mechanisms

Given that the rumen is the essential site of Mg²⁺ absorption under various feeding conditions, it has been proposed that both mechanisms work in parallel by ‘job sharing’ with an efficient uptake at all Mg²⁺ concentrations. At low ruminal Mg²⁺ concentrations, the PD-dependent and K⁺-sensitive mechanism might mediate Mg²⁺ transport with high affinity and low capacity. This became apparent in experiments by Ram et al. ⁽ Reference Ram, Schonewille and Martens ^{64 )} and Care et al. ⁽ Reference Care, Brown and Farrar ^{42 )}. High ruminal K⁺ intake reduced Mg²⁺ absorption to a higher extent at low ruminal Mg²⁺ concentration. Consequently, a possible negative effect of K⁺ intake will be pronounced at high ruminal K⁺ (>50 mmol/l) and low ruminal Mg²⁺ (<2 mmol/l) concentration (see below).

Vice versa, the PD-independent and K⁺-insensitive mechanism has a high capacity and low affinity and will thus primarily mediate transport at high Mg²⁺ (>3 mmol/l) concentrations. This uptake mechanism relies exclusively on the chemical gradients of the involved ions and will rise with increasing Mg²⁺ concentration (Table 1).

Table 1 Characteristics of magnesium transport across the rumen epithelium

PD_a, apical potential difference.

Mg²⁺ transport within the epithelium

The rumen epithelium is a squamous multilayered epithelium forming a functional syncytium comparable with the classical model of frog skin⁽ Reference Ussing and Zerahn ^{65 )}. Connections between cells of the various layers are formed by proteins such as connexin 43⁽ Reference Graham and Simmons ^{66 )}.

Basolateral extrusion

Mg²⁺ extrusion is related to the uptake of Na⁺. Reduction of serosal Na⁺ reduced J_ms Mg^{2+ (} Reference Leonhard-Marek and Martens ^{67 )} and in ruminal epithelial cells, the release or uptake of Mg²⁺ was dependent on the direction of the Na⁺ gradient⁽ Reference Schweigel, Vormann and Martens ^{68 )}. Furthermore, application of imipramine, an inhibitor of Na⁺/Mg²⁺ exchange, reduced Mg²⁺ transport⁽ Reference Leonhard-Marek, Stumpff and Brinkmann ^{12 ,} Reference Schweigel, Vormann and Martens ^{68 )}. The characterisation of Na⁺/Mg²⁺ exchange in HEK (human embryonic kidney) cells has revealed that the human gene SLC41A1 (solute carrier family 41 member 1) encodes for this Mg²⁺-transporting protein⁽ Reference Kolisek, Nestler and Vormann ^{69 ,} Reference Schweigel-Röntgen and Kolisek ^{70 )}. The Na⁺/Mg²⁺ exchanger is indirectly energised by Na⁺/K⁺-ATPase⁽ Reference Martens ^{43 )}. Although evidence for the extrusion of Mg²⁺ from giant squid axons via Na⁺/Mg²⁺ exchange had previously been obtained⁽ Reference Baker and Crawford ^{71 )}, ruminants were arguably the first mammalian species in which evidence for a (secondary) active epithelial Mg²⁺ transport could be obtained in an essential site of Mg²⁺ absorption (Fig. 2).

Fig. 2 Representation of transepithelial ruminal Mg²⁺ transport. The multi-layered epithelium is simplified to one compartment. Passive Mg²⁺ uptake is driven (1) mainly by the apical potential difference (PD_a) or (2) by the chemical gradient of the involved free ions. The PD-dependent uptake (1) is thought to involve homo- or heteromeric assemblies of the transient receptor potential channel proteins TRPM6 and TRPM7. The molecular identity of PD-independent (2) uptake is unknown. Basolateral extrusion occurs via Na⁺/Mg²⁺ exchange via solute carrier family 41 member 1 (SLC41A1). The negative effects of inhibitors (–) on various steps of Mg²⁺ transport are printed in italics. pJ_ms and J_sm represent the passive flow through the paracellular pathway. The cylindrical scheme represents a channel. PD_t, transepithelial potential difference; PD_b, basolateral potential difference; Mg²⁺ _i, intracellular ionised (free) Mg²⁺; DNP, 2,4-dinitrophenol; A^–, anion; C, carrier; P, pump (Na⁺/K⁺-ATPase). Example for PD_t (+15 mV)=PD_a (–45 mV) – PD_b (–60 mV). Depolarisation of PD_a by an increase of ruminal K⁺ increases PD_t.

Passive paracellular Mg²⁺ transport

The flux of Mg²⁺ from the serosal to the mucosal side (J_sm Mg²⁺) is entirely passive⁽ Reference Leonhard-Marek and Martens ^{48 )} with a permeability in the range of some 1×10^–6 cm/s. This low passive flow rate limits passive transport and is unimportant under in vivo conditions.

Saturation of Mg²⁺ transport

Mg²⁺ transport saturates in vitro ⁽ Reference Martens and Harmeyer ^{41 )} and in studies in vivo ⁽ Reference Care, Brown and Farrar ^{42 ,} Reference Martens ^{72 ,} Reference Martens ^{73 )} and probably includes the combined transport capacities of both uptake mechanisms. However, this saturation has never been observed in conventional balance studies. Weiss⁽ Reference Weiss ^{74 )} and Schonewille et al. ⁽ Reference Schonewille, Everts and Jittakhot ^{24 )} analysed Mg²⁺ intake and digestion in cows and found a linear correlation between Mg²⁺ intake and Mg²⁺ digestion, respectively. The observed saturation under experimental conditions simulated, but very likely did not represent, the real in vivo conditions⁽ Reference Care, Brown and Farrar ^{42 ,} Reference Martens ^{72 ,} Reference Martens ^{73 )}. Mg²⁺ was almost certainly ionised in these model studies and available for transport⁽ Reference Care, Brown and Farrar ^{42 ,} Reference Martens ^{72 ,} Reference Martens ^{73 )}. It is to be assumed that in the normal rumen fluid, Mg²⁺ is only partially ionised. Indeed, chelating Mg²⁺ by EDTA severely depresses Mg²⁺ transport⁽ Reference Leonhard, Smith and Martens ^{75 )}.

The classical implications of K⁺

High K⁺ intake significantly reduced Mg²⁺ digestion, plasma Mg²⁺ concentration and, consequently, urinary excretion in sheep⁽ Reference Fontenot, Miller and Whitehair ^{79 )} and cows⁽ Reference Schonewille, Van’t Klooster and Wouterse ^{80 )}. The reduced Mg²⁺ digestion was caused by a decrease in Mg²⁺ absorption and not by an increase in endogenous Mg²⁺ loss⁽ Reference Newton, Fontenot and Tucker ^{81 )}.

Meta-analysis of Mg²⁺ digestion: reduction by K⁺

The quantity of the effect of K⁺ on Mg²⁺ was analysed in a meta-analysis by Weiss⁽ Reference Weiss ^{74 )} in cows, yielding the following relationship:

(4) $$\eqalignno{ {\rm Digestible}\,{\rm Mg}^{{{\rm 2}{\plus}}} \, {\equals}\, &#x0026; {\rm 4}\!\cdot\!{\rm 5}\ \left( {{\rm {\scale70% S\scale70% E\scale70% M}}\,{\rm 4}\!\cdot\!0} \right){\rm g}/{\rm d}{\plus}0\!\cdot\!{\rm 24}\ \left( {\rm {\scale70% S\scale70% E\scale70% M}\,0\!\cdot\!0{\rm 7}} \right)\cr &#x0026; {\times}{\rm Mg}^{{{\rm 2}{\plus}}} \,{\rm intake}-{\rm 4}\!\cdot\!{\rm 4}\,{\rm g}/{\rm d}\left\ ( {{\rm {\scale70% S\scale70% E\scale70% M}}\,\,{\rm 2}\!\cdot\!{\rm 2}} \right){\times}{\rm K}^{{\plus}}\! ,$$

where digestible Mg²⁺ and Mg²⁺ intake are given in g/d, and K⁺ is given as % K⁺ in DM (thirty-nine diets, 162 cows).

Schonewille et al. ⁽ Reference Schonewille, Everts and Jittakhot ^{24 )} performed a second meta-analysis with a different set of experiments and with a larger number of diets and cows, yielding:

(5) $$\eqalignno{ {\rm Mg}^{{{\rm 2}{\plus}}} \left( {{\rm true}\,{\rm absorption}} \right)&#x0026;\, {\equals}\, {\rm 3}\!\cdot\!{\rm 6}\,{\rm g}/{\rm d}\ \left( {{\rm {\scale70% S\scale70% E\scale70% M}}\,0\!\cdot\!{\rm 67}} \right){\plus}0\!\cdot\!{\rm 2}\ \left( {{\rm {\scale70% S\scale70% E\scale70% M}}\,0\!\cdot\!0{\rm 1}} \right) \cr &#x0026; \quad {\times}{\rm Mg}^{{{\rm 2}{\plus}}}\,{\rm intake}-0\!\cdot\!0{\rm 8}\,{\rm g}/{\rm d }\left\ ( {{\rm {\scale70% S\scale70% E\scale70% M}}\,0\!\cdot\!0{\rm 14}} \right){\times}{\rm K}^{{\plus}}\! ,$$

where Mg²⁺ true absorption and intake are given in g/d, and K⁺ is given as g/kg in DM (sixty-eight diets, 323 cows).

True absorption can be transferred to apparent absorption (=digestible Mg²⁺) by correction for endogenous Mg²⁺ secretion (700 kg BW×4 mg/kg/d=2·8 g/d)^{( 86 ,} Reference Schonewille and Beynen ^{87 )}:

(6) $$\eqalignno {{\rm Mg}^{{{\rm 2}{\plus}}} \left( {{\rm apparent}\,{\rm absorption}} \right)\, {\equals}&#x0026;\, {\rm 3}\!\cdot\!{\rm 6}\,{\rm g}/{\rm d}-{\rm 2}\!\cdot\!{\rm 8}{\plus}0\!\cdot\!{\rm 2} \cr &#x0026;{\times}{\rm Mg}^{{{\rm 2}{\plus}}} \,{\rm intake}-0\!\cdot\!0{\rm 8}\,{\rm g}/{\rm d}{\times}{\rm K}^{{\plus}}\!;$$

(7) $$\eqalignno{{\rm Digestible}\,{\rm Mg}^{{{\rm 2}{\plus}}} \, {\equals}\, &#x0026;0\!\cdot\!{\rm 8}\,{\rm g}/{\rm d}{\plus}0\!\cdot\!{\rm 2}{\times}{\rm Mg}^{{{\rm 2}{\plus}}} \,{\rm intake} \cr &#x0026; -0\!\cdot\!0{\rm 8}\,{\rm g}/{\rm d}{\times}{\rm K}^{{\plus}}\!,$$

Where Mg²⁺ (apparent absorption), digestible Mg²⁺ and intake are given in g/d, and K⁺ is given in g/kg in DM.

At a K⁺ concentration of 1 % in the DM, the apparent Mg²⁺ digestion is slightly lower (20 %) than in the calculation of Weiss⁽ Reference Weiss ^{74 )} (24 %). However, Mg²⁺ digestion is more depressed at low Mg²⁺ intake.

The linear decrease in digestible Mg²⁺ with rising ruminal K⁺ (equations 4 and 7) is in contradiction to the discussed reduction of an effect of K⁺ at a higher Mg²⁺ intake. The major reason is probably the experimental design. The experiments of Ram et al. ⁽ Reference Ram, Schonewille and Martens ^{64 )} and Martens et al. ⁽ Reference Martens, Heggemann and Regier ^{58 )} were performed under identical conditions. Equations 4 and 5 are the result of meta-analyses of many balance studies.

The role of Na⁺

Insufficient Na⁺ intake releases aldosterone and decreases Na⁺ in both saliva and rumen fluid, while K⁺ is increased⁽ Reference Bailey ^{88 –} Reference Martens, Kubel and Gäbel ^{90 )}. Accordingly, Na⁺ deficiency in sheep caused a decrease of Na⁺ in saliva and rumen fluid, an increase of K⁺ in both liquids, and an enhanced PD_t, while Mg²⁺ absorption from the rumen decreased (see Table 2)⁽ Reference Martens, Kubel and Gäbel ^{90 )}. All of these changes were abolished by repletion of Na⁺. Furthermore, intravenous infusion of aldosterone in sheep caused an increase in K⁺ and a decrease in the Na⁺ concentration in the rumen. Concomitantly, ruminal Mg²⁺ concentration rose, while plasma Mg²⁺ declined⁽ Reference Charlton and Armstrong ^{91 )}. Since aldosterone alone does not change Mg²⁺ absorption⁽ Reference Martens and Hammer ^{92 )}, these effects are best explained by the aldosterone-induced elevation of the ruminal K⁺ concentration.

Table 2 Na⁺ deficiency and high K⁺ intake change the same rumen parameters and have identical effects on Mg²⁺ absorption

PD_t, transepithelial potential difference; ↑, increase; ↓, decrease.

Notably, K⁺ concentration in saliva can reach some 100 mmol/l in Na⁺-deficient animals. Assuming a salivary flow rate of 200 litres/d, this leads to a total influx of some 780 g K⁺/d and presents a significant risk for reduced Mg²⁺ absorption. The condition is easily overlooked, because overt clinical signs of Na⁺ deficiency are usually missing and because the large Na⁺ pool in the rumen can be mobilised to cover deficiency for a long time⁽ Reference Kemp and Geurink ^{93 )}. Furthermore, as the K⁺ concentration in the rumen fluid increases, the absorption of Na⁺ is enhanced⁽ Reference Lang and Martens ^{85 )}, which may help to compensate for Na⁺ deficiency.

Young spring grass frequently contains extremely low concentrations of Na^{+ (} Reference Morris and Gartner ^{94 )} and was suggested as a risk factor as early as 1966 by Metson et al. ⁽ Reference Metson, Saunders and Collie ^{95 )}: ‘If low sodium is confirmed as yet another stress factor in the development of hypomagnesaemia, most of the present analyses [of grass] would undoubtedly qualify as tetany prone’. This suggestion is in agreement with the observation of Butler⁽ Reference Butler ^{96 )} about a negative relationship between the low Na⁺ content of grass and the incidence of tetany. Vice versa, grass tetany caused by Na⁺ deficiency can be prevented by supplementation with NaCl⁽ Reference Paterson and Crichton ^{97 )}.

Protein and ammonia

Tetany-prone young grass in spring exhibits a high concentration of crude protein⁽ Reference Sjollema ^{14 )}, that causes an increase of up to 70 mmol/l ruminal ammonia⁽ Reference Annison, Lewis and Lindsay ^{98 )} and is associated with grass tetany⁽ Reference Kemp ^{99 )}. (The term ammonia is used without discrimination between NH₃ and NH₄ ⁺. Chemical symbols are used when a specification is required.) Relationships between ammonia and Mg²⁺ absorption have been tested with contradictory results: both inhibition of Mg²⁺ absorption and no effect on Mg²⁺ digestion at high ruminal ammonia, depending on the experimental conditions. A decrease in Mg²⁺ absorption was observed at a sudden increase in ruminal NH₄ ⁺ concentration. Intraruminal application of large amounts of ammonium acetate in cows caused a decrease both in plasma Mg²⁺ concentration and urinary Mg²⁺ excretion⁽ Reference Head and Rook ^{76 )}. When working with sheep⁽ Reference Martens and Rayssiguier ^{34 )} or young heifers⁽ Reference Martens, Heggemann and Regier ^{58 )}, respectively, Mg²⁺ absorption from the temporarily isolated rumen was severely reduced by increasing NH₄ ⁺ concentrations which agrees with studies of the rumen pouch⁽ Reference Care, Brown and Farrar ^{42 )}.

However, alterations in Mg²⁺ metabolism were not observed in chronic experiments with a delay in sampling after raising ruminal NH₄ ⁺ concentrations⁽ Reference Moore, Fontenot and Webb ^{100 ,} Reference Gäbel and Martens ^{101 )}. These observations led to the hypothesis that an acute increase in ruminal NH₄ ⁺ reduces Mg²⁺ absorption, but that when ruminal NH₄ ⁺ remains elevated for a period of days, an adaptational response normalises Mg²⁺ absorption. Gäbel & Martens⁽ Reference Gäbel and Martens ^{101 )} tested this hypothesis in vivo. Acute addition of artificial rumen fluid with 40 mmol NH₄ ⁺/l into the isolated sheep rumen significantly reduced Mg²⁺ absorption. In balance experiments, ruminal NH₄ ⁺ was rapidly increased from 4·81 (sd 0·18) to 47·9 (sd 3·1) mmol/l within 1 d. Mg²⁺ excretion in urine transiently decreased from 385 to 255 mg/d over 2 d, but on the 3rd day, urinary Mg²⁺ increased and returned to control values, despite high ruminal NH₄ ⁺ (36·1 (sd 4·8) mmol/l). Obviously, a sudden change in N intake and NH₄ ⁺ concentration impairs Mg²⁺ absorption, but adaptation occurred within 3 d.

The reason(s) for the temporary reduction of Mg²⁺ absorption by NH₄ ⁺ have not been studied. Ammonia is transported across the rumen epithelium both as NH₃ and NH₄ ⁺, depending on the pH⁽ Reference Abdoun, Stumpff and Wolf ^{102 )}. At a (physiological) pH of<7·0, NH₄ ⁺ is predominantly transported across cation channels in the apical membrane⁽ Reference Rosendahl, Braun and Schrapers ^{10 ,} Reference Abdoun, Stumpff and Wolf ^{102 )}, decreasing PD_a ⁽ Reference Lu, Stumpff and Deiner ^{103 )} and increasing PD_t ⁽ Reference Gäbel and Martens ^{101 )}. Since a variable fraction of the NH₄ ⁺ that is taken up is extruded in the form of NH₃, protons are released and decrease the cytosolic pH⁽ Reference Rosendahl, Braun and Schrapers ^{10 ,} Reference Lu, Stumpff and Deiner ^{103 )}, which stimulates apical Na⁺/H⁺ exchange and Na⁺ transport⁽ Reference Abdoun, Stumpff and Wolf ^{102 )}. These intraepithelial alterations of PD_a and intracellular pH (pH_i) offer some suggestions. Firstly, PD_a changes by some 10 mV when NH₄ ⁺ is elevated to 40 mmol/l⁽ Reference Lu, Stumpff and Deiner ^{103 )} which may inhibit channel-mediated uptake in analogy to what has been discussed for K⁺. However, interactions between pH_i and Mg²⁺ transport are a further possibility. Thus, the enhanced uptake of Na⁺ due to stimulation of Na⁺/H⁺ exchange⁽ Reference Abdoun, Stumpff and Wolf ^{102 )} should elevate cytosolic Na⁺ (Na⁺ _i), which can be expected to interfere with basolateral extrusion of Mg²⁺ via Na⁺/Mg²⁺ exchange. The possible mechanisms of adaptation are still unclear.

Ruminal pH

Only unbound Mg²⁺ in solution is available for transport across the ruminal epithelium and, accordingly, chelating Mg²⁺ by EDTA strongly reduces Mg²⁺ transport⁽ Reference Leonhard, Smith and Martens ^{75 )}. The range of free Mg²⁺ in the ruminal fluid varies from 34 to 77 % of the total amount⁽ Reference Grace, Caple and Care ^{104 ,} Reference Dalley, Isherwood and Sykes ^{105 )} and depends on various factors⁽ Reference Johnson, Helliwell and Jones ^{78 ,} Reference Dalley, Isherwood and Sykes ^{105 ,} Reference Xin, Tucker and Hemken ^{106 )}. One major factor determining the digestion of Mg²⁺ is the particle size of MgO⁽ Reference Xin, Tucker and Hemken ^{106 )}. Furthermore, free Mg²⁺ concentration in the rumen depends on pH⁽ Reference Dalley, Isherwood and Sykes ^{105 )}. The curvilinear relationship between rumen pH and Mg²⁺ solubility exhibits a steep slope between pH 5 and 7, which varies with diet⁽ Reference Dalley, Isherwood and Sykes ^{105 ,} Reference Horn and Smith ^{107 ,} Reference Johnson and Aubrey Jones ^{108 )} (Fig. 3). Most likely, increasing pH leads to the deprotonation of anionic binding sites in the ingested matter which are then available for binding of Mg²⁺. An enhancing effect of low ruminal pH on Mg²⁺ digestion was suggested early on by Wilcox & Hoff⁽ Reference Wilcox and Hoff ^{109 )}, and is probably related to an increase in unbound Mg²⁺. Horn & Smith⁽ Reference Horn and Smith ^{107 )} found a close and negative relationship between rumen pH and Mg²⁺ absorption before the duodenum. The obvious effect of pH on ionised Mg²⁺ is very likely the major reason for the influence of the diet on Mg²⁺ absorption, particularly with regard to carbohydrates. A causal relationship cannot be deduced from these studies, but the pH determines Mg²⁺ solubility with consequences for transport.

Fig. 3 Scheme of Mg²⁺ solubility in rumen fluid (redrawn from Dalley et al. ⁽ Reference Dalley, Isherwood and Sykes ^{105 )}). The slope of Mg²⁺ solubility between pH 5 and 7 is influenced by the diet (←, →).

There is also reason to believe that Mg²⁺-transporting proteins may be affected directly by changes in pH. Thus, patch clamp studies demonstrate that the conductance of monovalent cations is enhanced by a low pH in cells overexpressing TRPM6 and TRPM7⁽ Reference Li, Jiang and Yue ^{110 )}. At present it is unclear if Mg²⁺ conductance is similarly affected. Conversely, an acidic pH has been shown to decrease the expression of TRPM6 and other Mg²⁺-transporting proteins⁽ Reference Nijenhuis, Renkema and Hoenderop ^{111 )}, which probably contributes to the renal Mg²⁺ wasting that is observed in metabolic acidosis in man⁽ Reference Houillier ^{21 ,} Reference Dai, Friedman and Quamme ^{112 )}. Interestingly, chronic usage of proton pump inhibitors impairs gastrointestinal Mg²⁺ absorption⁽ Reference Atkinson, Reynolds and Travis ^{113 )}. The possible role of ruminal pH in the aetiology of grass tetany is not clear, because both higher⁽ Reference Horn and Smith ^{107 )} or lower pH has been reported⁽ Reference Balch and Rowland ^{114 )}. However, the close inverse correlation between ruminal pH and Mg²⁺ absorption before the duodenum⁽ Reference Horn and Smith ^{107 )} suggests that a high ruminal pH interferes with Mg²⁺ digestion, particularly at low DM intake in cold weather (H Meyer, personal communication) or as a consequence of pre-existing subclinical hypomagnesaemia with plasma Mg²⁺ concentration ≤0·8 mmol/l and no visible clinical signs such as ataxia or muscle spasms.

Mg²⁺ absorption and readily fermentable carbohydrates

A low level of fermentable carbohydrates in tetany-prone grass has been suggested to decrease Mg²⁺ availability⁽ Reference Metson, Saunders and Collie ^{95 )}. Vice versa, drenching of grazing dairy cattle with a starch solution increased plasma Mg²⁺ concentration⁽ Reference Wilson, Reid and Molloy ^{115 )} and digestion of Mg^{2+ (} Reference Giduck and Fontenot ^{57 )} although Mg²⁺ absorption was not consistently improved⁽ Reference House and Mayland ^{116 )}. In ruminal fluid, the addition of fermentable carbohydrates causes: (a) an increase in the concentration of SCFA⁽ Reference Giduck, Fontenot and Rahnema ^{117 )}, (b) a decrease in pH⁽ Reference Giduck, Fontenot and Rahnema ^{117 )}, which (c) enhances Mg²⁺ solubility⁽ Reference Dalley, Isherwood and Sykes ^{105 )}, (d) a decrease in NH₄ ⁺ concentration, and (e) an increase of the number and size of rumen papilla⁽ Reference Martens, Rabbani and Shen ^{118 )}, with the latter increasing the area for Mg²⁺ absorption⁽ Reference Gäbel, Martens and Suendermann ^{119 )}. Hence, Mg²⁺ digestion was enhanced in sheep by lactose⁽ Reference Rayssiguier and Poncet ^{120 )}.

The exact mechanism of the stimulation of Mg²⁺ transport by SCFA or HCO₃ ^–/CO₂ is not clear⁽ Reference Leonhard-Marek ^{60 )}. Notably, addition of fermentable carbohydrates to the diet with production of SCFA enhanced Mg²⁺ absorption from the caecum of rats⁽ Reference Demigné, Rémésy and Rayssiguier ^{121 )} and, in mice, inulin increased Mg²⁺ absorption and expression of TRPM6 and TRPM7 in the hindgut⁽ Reference Rondon, Rayssiguier and Mazur ^{122 )}. In studies with goats, Schonewille et al. ⁽ Reference Schonewille, Ram and Van’t Klooster ^{123 )} have demonstrated that the depressive effect of K⁺ can be compensated for by the addition of fermentable carbohydrates. Various reasons for this are conceivable. Influx of protonated SCFA with subsequent dissociation can be expected to lead to cell swelling, which, in turn, enhances monovalent currents both in cells hyperexpressing TRPM7 channels⁽ Reference Bessac and Fleig ^{124 )} and in native ruminal epithelial cells⁽ Reference Stumpff, Martens and Bilk ^{62 ,} Reference Georgi, Rosendahl and Ernst ^{63 )}. However, the most likely hypothesis is that the PD-independent pathway is stimulated by SCFA. Replacement of SCFA by gluconate significantly reduced the J_ms flux of Mg²⁺ and reduced uptake into cells⁽ Reference Leonhard-Marek, Gäbel and Martens ^{59 ,} Reference Schweigel and Martens ^{61 )}. This reduction in Mg²⁺ transport does not reflect binding by gluconate, because gluconate only weakly binds Mg^{2+ (} Reference Stumpff and McGuigan ^{125 )}, and does not affect the epithelial transport of Mg^{2+ (} Reference Leonhard-Marek, Marek and Martens ^{126 )}.

Mg²⁺ intake and digestion

The meta-analyses of Weiss⁽ Reference Weiss ^{74 )} and Schonewille et al. ⁽ Reference Schonewille, Everts and Jittakhot ^{24 )} demonstrated a linear correlation between Mg²⁺ intake and digestible Mg²⁺, suggesting a constant rate of Mg²⁺ absorption with no adaptation. However, McAleese et al. ⁽ Reference McAleese, Bell and Forbes ^{127 )} orally dosed ²⁸Mg²⁺ in sheep and observed a higher ²⁸Mg²⁺ absorption at deficient Mg²⁺ intake. In line with these findings are the results of Schweigel et al. ⁽ Reference Schweigel, Kolisek and Nikolic ^{52 ,} Reference Schweigel, Kuzinski and Deiner ^{56 )}: incubation of isolated rumen epithelial cells in a low- or high-Mg²⁺ medium caused a corresponding increase or decrease of in- and efflux mechanisms of Mg²⁺. Although the expression of TRPM7 was only slightly altered, both the expression of the Na⁺/Mg²⁺ exchanger⁽ Reference Schweigel, Kolisek and Nikolic ^{52 ,} Reference Schweigel, Kuzinski and Deiner ^{56 ,} Reference Schweigel, Park and Etschmann ^{128 )}, corresponding to SLC41⁽ Reference Schweigel-Röntgen and Kolisek ^{70 )}, and the Mg²⁺ channel MagT1 increased significantly at low Mg²⁺ incubation and vice versa⁽ Reference Schweigel, Kolisek and Nikolic ^{52 )}, supporting the assumption of the adaptation of Mg²⁺ transport at low Mg²⁺.

Allsop & Rook⁽ Reference Allsop and Rook ^{129 )} suggested that Mg²⁺ absorption is suppressed after increasing plasma Mg²⁺ concentration by intravenous infusion and concluded that ‘the most probable major site of action is therefore on the uptake of Mg from the reticulorumen’. Martens & Stössel⁽ Reference Martens and Stössel ^{23 )} tested this hypothesis and measured Mg²⁺ absorption from the isolated rumen in sheep. Mg²⁺ (net) transport was not influenced by increased plasma Mg²⁺ concentration or after 5 weeks of hypomagnesaemia, which is in contrast to the suggestion of McAleese et al. ⁽ Reference McAleese, Bell and Forbes ^{127 )} and can probably be explained by the method used: sheep were orally dosed with equal amounts of ²⁸Mg²⁺, and the appearance of the isotope in blood was taken as Mg²⁺ absorption. However, it is highly likely that the ratio between the radioactive isotope (²⁸Mg²⁺) and the total concentration of Mg²⁺ was much higher in Mg²⁺-deficient sheep than in controls. Accordingly, a higher absorption of ²⁸Mg²⁺ into blood could be expected even without a change of the total rate of Mg²⁺ – a possibility that was not considered by the authors since absorption from the rumen was not known at that time.

Endogenous Mg²⁺ secretion

Storry⁽ Reference Storry ^{26 )} made an estimation of Mg²⁺ secretion in various secretions of sheep (saliva, gastric juice, bile, etc.) and estimated a daily secretion of 192 mg/d in a 40 kg sheep or 4·8 mg/kg (live weight),with similar secretion rates of 3·4 and 5·04 mg/kg found by Care⁽ Reference Care ^{130 )}. A significant part of the endogenous Mg²⁺ loss is related to high flow rates of saliva. In sheep, Dua & Care⁽ Reference Dua and Care ^{131 )} estimated a secretion of about 40 % of the Mg²⁺ amount in the ECS or 2–3 mg/kg (live weight). This involuntary endogenous loss of Mg²⁺ is not constant. In sheep on an artificial, low-Mg²⁺ diet, secretion dropped to 0·4–1·4 mg/kg⁽ Reference Allsop and Rook ^{129 )}, which is probably related to the linear correlation between plasma Mg²⁺concentration and endogenous secretion of Mg²⁺ into the gut in general⁽ Reference Allsop and Rook ^{132 )}, and into the small intestine⁽ Reference Martens ^{31 )} or the bile in particular⁽ Reference Care ^{130 )}.

Schonewille & Beynen⁽ Reference Schonewille and Beynen ^{87 )} summarised data for the endogenous Mg²⁺ secretion by dairy cows (within a range from 1·5 to 6·0 mg/kg) and proposed 4 mg/kg, a value that is also used by the Gesellschaft für Ernährungsphysiologie (German Society for Nutritional Physiology)^{( 86 )}.

Animal breeds and Mg²⁺ absorption

The digestion of Mg²⁺ in cows⁽ Reference Greene, Baker and Hardt ^{133 )} and ruminal Mg²⁺ transport are influenced by animal breed⁽ Reference Leonhard-Marek, Marek and Martens ^{126 )}. Greene et al. ⁽ Reference Greene, Baker and Hardt ^{133 )} have shown that Mg²⁺ absorption is greater in Brahman than in Jersey, Holstein or Hereford cows. Leonhard-Marek et al. ⁽ Reference Leonhard-Marek, Marek and Martens ^{126 )} measured the net Mg²⁺ transport in vitro across isolated rumen epithelium of four breeds of sheep (Merino, Schwarzkopf, Skudde and Heidschnucke). Skudde transported significantly less Mg²⁺ under short-circuit conditions. The wide variation of Mg²⁺ digestion seen in different studies might have a genetic background and may contribute to heritability of Mg²⁺ in plasma⁽ Reference Tsiamadis, Banos and Panousis ^{16 )}. The significance of a genetic variation of Mg²⁺ transport proteins has been shown in man, where mutation of TRPM6 channels reduced transcellular Mg²⁺ transport in the intestine and kidney⁽ Reference Hoenderop and Bindels ^{50 )}.

Vitamin D and Mg²⁺ homeostasis

PTH and vitamin D₃ are the principal regulators of Ca²⁺ metabolism. Interactions between PTH, calcitriol and Mg²⁺ in cows are well established⁽ Reference Goff, Littledike and Horst ^{19 ,} Reference Schneider, Parkinson and Houston ^{134 ,} Reference Moate, Schneider and Leaver ^{135 )}, but the results are, in some cases, contradictory⁽ Reference Littledike and Goff ^{136 ,} Reference Hardwick, Jones and Brautbar ^{137 )}. Calcitriol increased plasma Ca²⁺ and Mg²⁺ concentrations in hypomagnesaemic sheep⁽ Reference Schneider, Parkinson and Houston ^{134 )} and Ca²⁺ concentration in cows, but decreased Mg²⁺ concentration⁽ Reference Moate, Schneider and Leaver ^{135 ,} Reference Sansom, Allen and Davies ^{138 )}. A calcitriol-dependent uptake of Mg²⁺ into soft tissue has been suggested⁽ Reference Moate, Schneider and Leaver ^{135 ,} Reference Yano, Horiuchi and Matsui ^{139 )}. Calcitriol did not change faecal excretion in cattle⁽ Reference Moate, Schneider and Leaver ^{135 )}, although calcitriol increased Mg²⁺ absorption from the rumen in sheep⁽ Reference Beardsworth ^{140 )}.

The infusion of bovine PTH in cows caused an increase in 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), Ca²⁺ and Mg²⁺ in plasma and a decrease in Mg²⁺ in urine⁽ Reference Goff, Littledike and Horst ^{19 )}, indicating enhanced Mg²⁺ resorption in the kidney. Dua et al. ⁽ Reference Dua, Leonhard and Martens ^{141 )} observed a trend for increased Mg²⁺ absorption from the reticulo-rumen of sheep after the onset of PTH or PTH-related protein infusions.

While interactions between the PTH and 1,25(OH)₂D₃ axis and Mg²⁺ metabolism can thus be observed, the physiological significance of this interaction is not clear. The effect of 1,25(OH)₂D₃ on epithelial Ca²⁺ transport is classical and related to increased expression and activity of TRPV5 and TRPV6 channels⁽ Reference Hoenderop and Bindels ^{50 )}. These channels are non-selective cation channels with a high selectivity for Ca²⁺ over monovalent cations. However, a certain, albeit low, permeability to Mg²⁺ is to be expected. In summary, the possible stimulation of Mg²⁺ transport by 1,25(OH)₂D₃ or effects of PTH should be considered as a side-effect of Ca²⁺ homeostasis.

Ionophores and Mg²⁺ digestion

Ionophores like monensin and lasalocid significantly increase Mg²⁺ digestion⁽ Reference Greene, Schelling and Byers ^{142 )}. Both ionophores lowered ruminal K⁺ concentrations in steers, suggesting a diminution of the reduction of K⁺ on Mg²⁺ transport.

Mg²⁺ filtration

Plasma Mg²⁺ varies from 0·9 to 1·2 mmol/l. Some 60–80 % or 0·48–0·96 mmol/l of plasma Mg²⁺ is ultrafiltrable so that, on a daily basis, roughly 29–59 g Mg²⁺/d will appear in the glomerular filtrate of a cow of 650 kg BW (calculated with glomerular filtration rate (GFR) data of Murayama et al. ⁽ Reference Murayama, Miyano and Sasaki ^{148 )}). Possible effects of GFR on Mg²⁺ filtration are not known.

Urinary Mg²⁺ excretion

The adaptation of renal Mg²⁺ transport activity in cows to various levels of intake has been illustrated by Schonewille⁽ Reference Schonewille, van‘t Klooster and Wouterse ^{168 )} and Holtenius et al. ⁽ Reference Holtenius, Kronqvist and Briland ^{169 )}. Urinary excretion of Mg²⁺ rises in a quasi-exponential manner with plasma Mg²⁺ concentration. However, urinary Mg²⁺ drops rapidly with falling plasma Mg²⁺, but levels off at 0·61–0·73 mmol/l, after which Mg²⁺ almost ceases to be excreted in urine⁽ Reference Storry and Rook ^{170 )}. Accordingly, a dairy cow with a plasma Mg²⁺ concentration<0·8 mmol/l has to be considered at risk of hypomagnesaemia. This range of Mg²⁺ concentration appears to be a threshold. In a recent meta-analysis of Mg²⁺ metabolism in man, a concentration of ≥0·87 mmol/l leads to substantial urinary Mg²⁺ excretion⁽ Reference Zhang, Del Gobbo and Hruby ^{171 )}.

Urinary Mg²⁺ excretion is a more sensitive indicator of Mg²⁺ availability than the plasma concentration. Rook & Balch⁽ Reference Rook and Balch ^{172 )} observed a much more pronounced decline of Mg²⁺ in urine than in plasma following a change in diet. The tight control of Mg²⁺ transport activity, particularly in the DCT⁽ Reference Houillier ^{21 )} but also in the TAL⁽ Reference Efrati, Hirsch and Kladnitsky ^{153 )}, explains these classical observations.

The adjustment of renal Mg²⁺ excretion to changes in dietary intake with altered Mg²⁺ absorption (influx) not only ensures the maintenance of Mg²⁺ homeostasis in most feeding situations (Fig. 1), but also provides the practitioner with a diagnostic tool. According to the data of Kemp⁽ Reference Kemp ^{173 )}, Mg²⁺ influx can be considered to be sufficient at urinary Mg²⁺>4·4 mmol/l, while a range of 0·87–4·4 mmol/l might indicate a risk of Mg²⁺ shortage. Urinary Mg²⁺<1 mmol/l is probably a reliable indicator of insufficient intake/absorption.

Interaction of magnesium and calcium

Mutual interactions of transport between Ca²⁺ and Mg²⁺ have been observed⁽ Reference Quamme ^{174 )}. Hypercalcaemia caused a large increase in urinary Mg²⁺ excretion. Vice versa and again in rats, infusion of Mg²⁺ caused an increase of urinary Ca²⁺ associated with a reduction in Ca²⁺ uptake via TRPV5⁽ Reference Bonny, Rubin and Huang ^{175 )}. A mutual interaction of Ca²⁺ and Mg²⁺ has also been found in cows, with negative interactions observed both on the level of the kidney⁽ Reference Roche, Morton and Kolver ^{176 )} and the rumen⁽ Reference Care, Brown and Farrar ^{42 ,} Reference Oehlschlaeger, Wilkens and Schroeder ^{177 )}.

Plasma Mg²⁺ and tetany

Sjollema⁽ Reference Sjollema ^{13 ,} Reference Sjollema ^{14 )} first demonstrated the relationship between the clinical symptoms of grass tetany and hypomagnesaemia. However, the Mg²⁺ concentration in the plasma of afflicted animals exhibits some variation (Table 3), and the severity of the nervous disturbances is not closely related to the plasma Mg²⁺ concentration⁽ Reference Halse ^{184 )}. Possibly, the speed of plasma Mg²⁺ decline promotes the onset of clinical manifestations⁽ Reference Allcroft and Burns ^{185 )}.

Table 3 Status of Mg²⁺ metabolism and plasma Mg²⁺ concentration

At values below 0·9 mmol/l, both an adequate supply of Mg²⁺ or impending clinical hypomagnesaemia are possibilities, so that a safe assessment of Mg²⁺ status should involve a determination of urinary Mg²⁺ excretion. Even then, difficulties in judging Mg²⁺ status can be clearly seen in a study involving non-pregnant lactating cows with normal Mg²⁺ intake (29–32·5 g/d) and plasma Mg²⁺ concentration of 0·75–1·1 mmol/l⁽ Reference Schweigel, Voigt and Mohr ^{186 )}. After intravenous infusion of Mg²⁺ and despite a slight increase in plasma Mg²⁺ in four of the nine animals, the fractional renal Mg²⁺ excretion decreased, indicating Mg²⁺ retention after the Mg²⁺ load and pointing towards a possible Mg²⁺ deficit. Despite these uncertainties, low plasma Mg²⁺ concentrations almost invariably precede the onset of neurological symptoms with impaired function of the CNS.

Subclinical hypomagnesaemia

It is important to note that the appearance of clinically relevant neurological symptoms is not obligatory, even when plasma levels of Mg²⁺ are low. Hypomagnesaemia of about 0·5 mmol/l was induced in sheep by feeding a low-Mg²⁺ diet for 5 weeks without appearance of any neurological symptoms⁽ Reference Martens and Stössel ^{23 )}. It is very likely that Mg²⁺ concentration in the CSF is maintained when the induction of hypomagnesaemia with a low-Mg²⁺ diet is gradual (see above). It should be noted that even in the absence of clear neurological symptoms, animals may suffer from various non-neurological manifestations of hypomagnesaemia.

Interactions between hypomagnesaemia and the regulation of Ca²⁺ metabolism were observed early on. Thus, Allen et al. ⁽ Reference Allen, Sansom and Davies ^{198 )} showed a correlation between subclinical hypomagnesaemia and the occurrence of milk fever with plasma Mg concentrations of<0·8 mmol/l. Subclinical hypomagnesaemia has a negative effect on the release of PTH⁽ Reference Littledike and Goff ^{136 ,} Reference Anast, Mohs and Kaplan ^{199 ,} Reference Rayssiguier, Garel and Davicco ^{200 )}, the functioning of PTH on the target organ⁽ Reference MacManus, Heaton and Lucas ^{201 ,} Reference Goff ^{202 )} and the conversion of 25(OH)D₃ to 1,25(OH)₂D₃ (calcitriol)⁽ Reference Horsting and DeLuca ^{203 )}. Moreover, in organ cultures of fetal rat bone, the release of Ca by supplementing 1,25(OH)₂D₃ or PTH was reduced at low (<0·8 mmol/l) Mg concentration⁽ Reference Johannesson and Raisz ^{204 )}. Furthermore, regulation of Ca homeostasis was found to deteriorate with induction of secondary hypocalcaemia in calves with hypomagnesaemia⁽ Reference Rayssiguier, Garel and Davicco ^{200 )}. These results correspond very well with in vivo observations of Sansom et al. ⁽ Reference Sansom, Manston and Vagg ^{205 )}, who found that the mobilisation of Ca from bone was lowered significantly in cows with hypomagnesaemia. A subsequent study by van de Braak et al. ⁽ Reference van de Braak, van’t Klooster and Malestein ^{206 )} confirmed these findings.