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Magnesium homeostasis in cattle: absorption and excretion

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  • Health and Medical University Potsdam

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Magnesium (Mg ²⁺ ) is an essential mineral without known specific regulatory mechanisms. In ruminants, plasma Mg ²⁺ concentration depends primarily on the balance between Mg ²⁺ absorption and Mg ²⁺ excretion. The primary site of Mg ²⁺ absorption is the rumen, where Mg ²⁺ is apically absorbed by both potential-dependent and potential-independent uptake mechanisms, reflecting involvement of ion channels and electroneutral transporters, respectively. Transport is energised in a secondary active manner by a basolateral Na ⁺ /Mg ²⁺ exchanger. Ruminal transport of Mg ²⁺ is significantly influenced by a variety of factors such as high K ⁺ concentration, sudden increases of ammonia, pH, and the concentration of SCFA. Impaired Mg ²⁺ absorption in the rumen is not compensated for by increased transport in the small or large intestine. While renal excretion can be adjusted to compensate precisely for any surplus in Mg ²⁺ uptake, a shortage in dietary Mg ²⁺ cannot be compensated for either via skeletal mobilisation of Mg ²⁺ or via up-regulation of ruminal absorption. In such situations, hypomagnesaemia will lead to decrease of a Mg ²⁺ in the cerebrospinal fluid and clinical manifestations of tetany. Improved knowledge concerning the factors governing Mg ²⁺ homeostasis will allow reliable recommendations for an adequate Mg ²⁺ intake and for the avoidance of possible disturbances. Future research should clarify the molecular identity of the suggested Mg ²⁺ transport proteins and the regulatory mechanisms controlling renal Mg excretion as parameters influencing Mg ²⁺ homeostasis.
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Magnesium homeostasis in cattle: absorption and excretion
Holger Martens
1
*, Sabine Leonhard-Marek
2
, Monika Röntgen
3
and Friederike Stumpff
1
1
Institute for Veterinary Physiology, Freie Universität Berlin, Berlin, Germany
2
Department of Physiology, University of Veterinary Medicine, Foundation, Hannover, Germany
3
Institute of Muscle Biology and Growth, Leibniz Institute for Farm Animal Biology (FBN), 18196 Dummerstorf, Germany
Abstract
Magnesium (Mg
2+
) is an essential mineral without known specic regulatory mechanisms. In ruminants, plasma Mg
2+
concentration depends
primarily on the balance between Mg
2+
absorption and Mg
2+
excretion. The primary site of Mg
2+
absorption is the rumen, where Mg
2+
is apically
absorbed by both potential-dependent and potential-independent uptake mechanisms, reecting involvement of ion channels and electroneutral
transporters, respectively. Transport is energised in a secondary active manner by a basolateral Na
+
/Mg
2+
exchanger. RuminaltransportofMg
2+
is
signicantly inuenced by a variety of factors such as high K
+
concentration, sudden increases of ammonia, pH, and the concentration of SCFA.
Impaired Mg
2+
absorption in the rumen is not compensated for by increased transport in the small or large intestine. While renal excretion can be
adjusted to compensate precisely for any surplus in Mg
2+
uptake, a shortage in dietary Mg
2+
cannot be compensated for either via skeletal
mobilisation of Mg
2+
or via up-regulation of ruminal absorption. In such situations, hypomagnesaemia will lead to decrease of a Mg
2+
in the
cerebrospinal uid and clinical manifestations of tetany. Improved knowledge concerning the factors governing Mg
2+
homeostasis will allow reliable
recommendations for an adequate Mg
2+
intake and for the avoidance of possible disturbances. Future research should clarify the molecular identity
of the suggested Mg
2+
transport proteins and the regulatory mechanisms controlling renal Mg excretion as parameters inuencing Mg
2+
homeostasis.
Key words: Rumen: Epithelial transport: Tetany: Cows: Kidneys
Introduction
Magnesium (Mg
2+
) is an essential mineral
(1)
and its binding is of
central importance for enzymic reactions after combining with
the enzyme or substrate. The cytosolic concentration of the free,
ionised Mg
2+
ion is about 1 mmol/l, but the total intracellular
concentration is much higher since numerous anions such
as phosphate groups in nucleic acids or ATP
4
bind Mg
2+(2)
.
Furthermore, Mg
2+
acts as a modulator of synaptic transmission
in the central nervous system (CNS)
(3,4)
, at the motoric
endplate
(5)
, in immunological pathways
(6)
andintimekeeping
(7)
.
Importantly, Mg
2+
is involved in the gating of ion channels
(8)
.
Many transient receptor potential (TRP) channels are regulated by
Mg
2+
in a voltage-dependent manner
(9)
and are involved in the
transport of cations across the ruminal epithelium
(1012)
.
The modulation of channel function in the CNS by Mg
2+
is
probably the reason for neurological symptoms such as ataxia,
recumbency, convulsions, and nally tetanic muscle spasms in
hypomagnesaemia and has been well known in cattle for some
80 years as grass tetany
(13,14)
.
The present review attempts to outline the principles of
Mg
2+
homeostasis with particular emphasis on the site and
mechanism of Mg
2+
absorption, renal excretion and possible
imbalances such as tetany.
Magnesium homeostasis
Plasma magnesium
Despite the absence of a (known) specic regulatory system,
Mg
2+
in plasma is kept within the range of 0·91·2 mmol/l,
provided that inux (via absorption) into the extracellular space
(ECS) including plasma is larger than the efux (requirement
and excretion). In humans, six genomic regions have been
implicated in the maintenance of plasma Mg
2+
concentra-
tion
(15)
, and similar gene loci may explain the heritability of
plasma Mg
2+
concentration in dairy cows (0·200·43)
(16)
.
Plasma Mg
2+
is known to be inuenced in a non-specic
manner by catecholamines
(17)
, insulin
(18)
and parathyroid
hormone (PTH)
(19)
. In addition, epidermal growth factor has
recently been shown to have magnesiotropic effects via TRPM6
channels, which regulate renal and intestinal Mg
2+
absorption
(20)
.
Distribution of magnesium
Some 6070 % of total body Mg
2+
is bound in the skeleton.
A further 30 % is found in the intracellular space (ICS) but only
15 % of intracellular Mg
2+
is in the ionised form
(21)
. The Mg
2+
in the ECS only reects about 1 % of total body Mg
2+
.
Nutrition Research Reviews
*Corresponding author: Dr Holger Martens, email Holger.Martens@fu-berlin.de
Abbreviations: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; BW, body weight; CNNM, cyclin and CBS domain divalent metal cation transport mediator;
CNS, central nervous system; CSF, cerebrospinal uid; DCT, distal convoluted tubule; ECS, extracellular space; ICS, intracellular space; PD, potential difference;
PTH, parathyroid hormone; TAL, thick ascending limb of Henle; TRP, transient receptor potential.
Nutrition Research Reviews, page 1 of 17 doi:10.1017/S0954422417000257
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Between 20 and 40 % of plasma Mg
2+
is bound to albumin and
globulin and some 10 % complexes with small anions such as
citrate, phosphate and bicarbonate, so that 5070 % are ionised.
According to a classical estimate
(22)
, the total content of Mg
2+
within the body of calves can be calculated from:
Mg gðÞ=0655 ´BW ðkgÞðÞ35;(1)
where BW is body weight in kg.
Provided that a similar relationship holds for adult ruminants,
the total body Mg
2+
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 45 g would
be found in the total ECS.
Regulation of magnesium homeostasis
The range of plasma Mg
2+
primarily depends on the inux of
Mg
2+
from the gastrointestinal tract into the ECS (a) and on the
efux 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). Mg
2+
not required for bd is excreted into urine (e). Plasma
Mg
2+
concentration thus depends on the daily Mg
2+
balance and
is given by:
a=b+c+d+e;(2)
where a is Mg
2+
absorption (g/d) (inux), b is Mg
2+
efux in
milk (g/d), c is Mg
2+
uptake (efux) into the ICS (g/d), d is
intestinal Mg
2+
secretion (efux) (g/d), and e is renal Mg
2+
excretion (efux) (g/d).
A scheme of Mg
2+
metabolism for a cow with a BW of 700 kg
and a milk production of 40 kg/d is given in Fig. 1.
In an adult and not growing, non-pregnant cow, net uptake
of Mg
2+
into the ICS and bone at adequate Mg intake is
marginal, so that equation (2) can be simplied to:
a=b+d+e;(3)
where a is Mg
2+
absorption (g/d) (inux), b is Mg
2+
efux in
milk (g/d), d is endogenous Mg
2+
secretion (efux) (g/d), and e
is renal Mg
2+
excretion (efux) (g/d).
Because Mg
2+
absorption (a) rarely equals Mg
2+
efux,
additional mechanisms are necessary for the adjustment, which
is very efciently controlled by the kidneys at Mg
2+
surplus.
However, mobilisation from the skeleton or the cytosol is
very limited
(21)
. This suggests that Mg
2+
inux was very rarely
limited during evolution. Obviously, Mg
2+
intake and absorp-
tion (a) were generally above requirement (b + c + d), so that
an efcient renal excretion of the surplus was sufcient for the
regulation of Mg
2+
homeostasis (e). Moreover, Mg
2+
is not
very toxic, and hence transient hypermagnesaemia (rapid
inux >efux) is well tolerated
(23)
. Therefore, absorption from
the gastrointestinal tract is the key factor determining plasma
Mg
2+
levels, which can only be kept constant when the daily
requirement is replaced by an adequate absorption. A better
comprehension of the gastrointestinal absorption (inux) and
its large variation
(24)
appears to be a key factor for under-
standing Mg
2+
homeostasis.
Mg
2+
absorption from the ruminant gastrointestinal tract
Site of magnesium absorption
Early studies in vivo suggested the distal part of the small
intestine as the site of Mg
2+
absorption
(25)
and Storry
(26)
stated
that there is no evidence to assume that ... a mechanism exists
Nutrition Research Reviews
Oral Mg2+ intake:
50 g/d
Faecal Mg2+:
37·2 g/d + 2·8 g/d = 40 g/d
True
absorption
12·8 g/d
Extracellular pool:
4–5 g Mg2+
Milk 40 litres/d: 4·8 g/d
Urine: 5·2 g/d
Endog.
secretion
2·8 g/d
?
ICS
about
130 g
?
Bone
about
320 g
Fetus
Available Mg2+:
12·8 g/d
Fig. 1. Scheme of Mg
2+
metabolism in a non-pregnant dairy cow of 700 kg body weight (BW). The daily Mg
2+
intake is 50 g, with true Mg
2+
absorption being 12·8g/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 Mg
2+
digestion of
10 g/d (20 %). An amount of 14·8g Mg
2+
/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
(26)
. The
unidirectional flow of Mg
2+
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 0·2g Mg
2+
/d towards the fetus has to be included
(207)
.
2 H. Martens et al.
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for the transport of calcium and magnesium across the rumen
epithelium. In contrast, Harrison et al.
(27)
observed Mg
2+
absorption proximal to the duodenum and Marongiu from the
rumen uid
(28)
. In a later study, Rogers & vant Klooster
(29)
measured mineral ow rates along the gut and demonstrated
absorption before the duodenum. Martens
(30)
summarised
data from sheep and cows. The rate of absorption before
the duodenum increased linearly with Mg
2+
intake
(31)
. A net
secretion was observed into the small intestine; the same
amount was absorbed in the large intestine.
Regarding the location of Mg
2+
absorption before the duo-
denum, uptake from the omasum but not from the abomasum
was demonstrated
(32,33)
. However, Martens & Rayssiguier
(34)
showed that, in vitro, the transport capacity of the rumen
epithelium was large and predominant.
Physiological signicance of the rumen. An important
nding was that Mg
2+
absorption from the forestomachs was
essential for maintaining normal plasma Mg
2+
and a precondition
for Mg
2+
homeostasis
(35)
. Reduced Mg
2+
absorption from the
forestomach was not compensated for by absorption in the
intestine
(36)
.
Absorption from the large intestine.Mg
2+
absorption was
shown to switch from the hindgut to the developed rumen after
dietary transition from milk to solid feed in calves
(37)
and
lambs
(38)
.Mg
2+
absorption from the hindgut is maintained in
adult animals and can be used for the treatment of acute
hypomagnesaemia
(39)
.
Mechanism of ruminal Mg
2+
transport
Scott
(40)
analysed the passive driving forces across the
rumen epithelium and concluded that the chemical gradient
of Mg
2+
for passive movement from the rumen to plasma
was opposed by the stronger electrical gradient (blood side
positive 3060 mV), which thus prevented passive paracellular
uptake of the Mg
2+
ion from the rumen to blood. Accordingly,
Mg
2+
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
(4143)
showing: (a) net trans-
port of Mg
2+
from the rumen to blood; (b) saturation kinetics;
(c) signicantly lower transport at lower temperature; and
(d) reduced transport by inhibition of Na
+
/K
+
-ATPase (ouabain
or dinitrophenol). Furthermore, it was shown that bulk ow
could not explain ruminal Mg
2+
transport
(44)
, as had been
proposed for rats
(45)
.
The movement of Mg
2+
across the multilayered epithelium
includes: (a) uptake across the apical membrane; (b) the
passage of Mg
2+
across the various epithelial layers of the
multilayered epithelium; and (c) release across the basolateral
membrane. In cases where the passive gradients are sufcient,
transepithelial transfer may further involve (d) possible para-
cellular passive movement.
Epithelial mechanisms. For many decades, characterisation of
the transcellular transport pathway suffered from a lack of
knowledge about Mg
2+
transport. The existence of Mg
2+
ion
channels was still widely considered an unproven hypothesis.
However, transport mechanisms for other ions (for example, Na
+
or Ca
2+
) had been clearly established in other epithelia, such as in
rabbit ileum
(46)
. Simply put, transport of ions across epithelia is
either inuenced by the transepithelial potential difference (PD
t
)
or not. By varying the electrical driving force for a specicion
(ξ)
(46)
, and plotting the measured ux rate over ξ, it is possible to
differentiate between the PD-independent ux (given by the
y-intercept in the plot) and the PD-dependent ux (given by the
slope of the plotted curve).
Potential-dependent Mg
2+
uptake: The suggestion that Mg
2+
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 Mg
2+
transport
(36,47)
.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 (rum-
inal) 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 Mg
2+
uptake. Mucosal to serosal Mg
2+
transport rates (J
ms
Mg
2+
) revealed a linear correlation between
ξ(PD
t
) and J
ms
Mg
2+
within 25 and + 25 mV
(48)
. The obtained
slope conrmed the suggestion of PD-dependent J
ms
Mg
2+
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 Mg
2+
in the apical
membrane is supported by data from microelectrode experiments.
Leonhard-Marek & 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
(10)
.
In further ux measurements, Mg
2+
transport was reduced not
only by elevation of the PD
t
but also by the apical K
+
con-
centration
(47,48)
. Depolarisation of PD
a
by K
+
is the most likely
explanation for the reduced mucosal to serosal ux of Mg
2+
(J
ms
)
at high concentrations of K
+
(80 mmol/l) and argues for the uptake
of Mg
2+
by a PD-dependent mechanism. Since the ionised intra-
cellular Mg
2+
concentration (0·54 mmol/l)
(49)
is lower than the
concentration of Mg
2+
in the rumen, the uptake of Mg
2+
is driven
by the electrochemical gradient across the apical membrane.
The ruminal Mg
2+
channel: The signicant correlation
between changes of PD
a
and Mg
2+
transport led to the sug-
gestion of an apical Mg
2+
channel
(47)
, long before such channels
were cloned. Channel-mediated transport of Mg
2+
is now well
established
(50)
. Thus, hypomagnesaemia in man is now known
to be caused by the mutation of a channel of the TRP gene
family, TRPM6
(51)
. TRPM6 plays a key role in the intestinal and
renal absorption of Mg
2+
in mice
(50)
. Expression of mRNA
encoding for this protein by the rumen epithelium suggests a
similar role in the ruminal absorption of Mg
2+(10)
.
A further member of this channel family, TRPM7, has been
demonstrated in ruminal epithelial cells as mRNA
(10,52)
and
Nutrition Research Reviews
Magnesium in ruminants 3
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protein
(52)
and is thought to play a role in intracellular Mg
2+
homeostasis
(53)
. Since experiments in TRPM7-decient mice by
Ryazanova et al.
(54)
demonstrate disturbed intestinal Mg
2+
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 func-
tional Mg
2+
channel
(55)
. MagT1 is a further candidate gene for
the PD-dependent uptake pathway in ruminal epithelial
cells
(52,56)
.
Potential difference-independent (electroneutral) Mg
2+
uptake: In addition to the channel-mediated pathway, a second,
PD-independent Mg
2+
uptake pathway mediates Mg
2+
trans-
port
(48)
. The charge of Mg
2+
is compensated by co-transport
with anions or counter-transport of cations. Interestingly, the
intake of high levels of readily fermentable carbohydrates
(57)
increased Mg
2+
digestion. Furthermore, SCFA or CO
2
enhanced
ruminal Mg
2+
absorption in vivo
(58)
and stimulated J
ms
Mg
2+
in vitro
(59)
. Since both fermentation products acidify the epi-
thelium, Mg
2+
/2H
+
exchange has been proposed to represent
this transport mechanism
(59,60)
.
However, Schweigel & Martens
(61)
found no experimental
evidence for directly coupled Mg
2+
/2H
+
exchange in isolated
ruminal epithelial cells of sheep and suggested a co-transport of
Mg
2+
with an anion such as HCO
3
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
(62,63)
. This opens the possibility that the stimulation of
Mg
2+
by SCFA and CO
2
may not exclusively represent PD-
independent Mg
2+
transport but also involves stimulation of PD-
dependent mechanisms. Finally, the activity of the ruminal
vacuolar H
+
-ATPase modulates Mg
2+
transport
(61)
, possibly by
increasing PD
a
and thus enhancing the uptake of Mg
2+
. Such a
mechanism would represent functional albeit not xed Mg
2+
/H
+
exchange. Currently, neither the stoichiometry nor the mole-
cular identity of the PD-independent Mg
2+
transporter is known.
Physiological consequences of two uptake mechanisms:
Given that the rumen is the essential site of Mg
2+
absorption
under various feeding conditions, it has been proposed that
both mechanisms work in parallel by job sharingwith an
efcient uptake at all Mg
2+
concentrations. At low ruminal Mg
2+
concentrations, the PD-dependent and K
+
-sensitive mechanism
might mediate Mg
2+
transport with high afnity and low capa-
city. This became apparent in experiments by Ram et al.
(64)
and
Care et al.
(42)
. High ruminal K
+
intake reduced Mg
2+
absorption
to a higher extent at low ruminal Mg
2+
concentration. Con-
sequently, a possible negative effect of K
+
intake will be
pronounced at high ruminal K
+
(>50 mmol/l) and low ruminal
Mg
2+
(<2 mmol/l) concentration (see below).
Vice versa, the PD-independent and K
+
-insensitive mecha-
nism has a high capacity and low afnity and will thus primarily
mediate transport at high Mg
2+
(>3 mmol/l) concentrations.
This uptake mechanism relies exclusively on the chemical
gradients of the involved ions and will rise with increasing Mg
2+
concentration (Table 1).
Mg
2+
transport within the epithelium: The rumen epithelium
is a squamous multilayered epithelium forming a functional
syncytium comparable with the classical model of frog skin
(65)
.
Connections between cells of the various layers are formed by
proteins such as connexin 43
(66)
.
Basolateral extrusion: Mg
2+
extrusion is related to the uptake
of Na
+
. Reduction of serosal Na
+
reduced J
ms
Mg
2+(67)
and in
ruminal epithelial cells, the release or uptake of Mg
2+
was
dependent on the direction of the Na
+
gradient
(68)
. Furthermore,
application of imipramine, an inhibitor of Na
+
/Mg
2+
exchange,
reduced Mg
2+
transport
(12,68)
. The characterisation of Na
+
/Mg
2+
exchange in HEK (human embryonic kidney) cells has revealed
that the human gene SLC41A1 (solute carrier family 41 member 1)
encodes for this Mg
2+
-transporting protein
(69,70)
.TheNa
+
/Mg
2+
exchanger is indirectly energised by Na
+
/K
+
-ATPase
(43)
. Although
evidence for the extrusion of Mg
2+
from giant squid axons via
Na
+
/Mg
2+
exchange had previously been obtained
(71)
, ruminants
were arguably the rst mammalian species in which evidence for
a (secondary) active epithelial Mg
2+
transport could be obtained
in an essential site of Mg
2+
absorption (Fig. 2).
Passive paracellular Mg
2+
transport: The ux of Mg
2+
from
the serosal to the mucosal side (J
sm
Mg
2+
) is entirely passive
(48)
with a permeability in the range of some 1 ×10
6
cm/s. This low
passive ow rate limits passive transport and is unimportant
under in vivo conditions.
Saturation of Mg
2+
transport
Mg
2+
transport saturates in vitro
(41)
and in studies
in vivo
(42,72,73)
and probably includes the combined transport
capacities of both uptake mechanisms. However, this saturation
has never been observed in conventional balance studies.
Weiss
(74)
and Schonewille et al.
(24)
analysed Mg
2+
intake and
digestion in cows and found a linear correlation between Mg
2+
intake and Mg
2+
digestion, respectively. The observed satura-
tion under experimental conditions simulated, but very likely
did not represent, the real in vivo conditions
(42,72,73)
.Mg
2+
was
almost certainly ionised in these model studies and available for
transport
(42,72,73)
. It is to be assumed that in the normal rumen
Nutrition Research Reviews
Table 1. Characteristics of magnesium transport across the rumen epithelium
Luminal Mg
2+
uptake
Ions Driving force Properties Nomenclature Basolateral Mg
2+
extrusion
Mg
2+
Electrical gradient (PD
a
) High affinity
Low capacity
PD-dependent
K
+
-sensitive
Na
+
/Mg
2+
exchanger
Mg
2+
+ anions (?) Chemical gradient Low affinity
High capacity
PD-independent
K
+
-insensitive
Na
+
/Mg
2+
exchanger
PD
a
, apical potential difference.
4 H. Martens et al.
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uid, Mg
2+
is only partially ionised. Indeed, chelating Mg
2+
by
EDTA severely depresses Mg
2+
transport
(75)
.
Modulation of ruminal Mg
2+
transport
In one of his rst publications, Sjollema
(14)
reported the com-
position of tetany-prone grass with high concentrations of K
+
and N, low concentrations of Na
+
, while levels of Mg
2+
were
moderate but not low. Hence, this disease does not arise by
inadequate intake of Mg
2+
alone
(76)
. Hypomagnesaemic tetany
also occurred after changing the diet despite equal Mg
2+
con-
tent
(77)
, and a decrease in plasma Mg
2+
was observed even with
an increase in Mg
2+
intake
(78)
. Today, there can be no doubt
that various dietary factors interfere with Mg
2+
transport.
The classical implications of K
+
High K
+
intake signicantly reduced Mg
2+
digestion, plasma
Mg
2+
concentration and, consequently, urinary excretion in
sheep
(79)
and cows
(80)
. The reduced Mg
2+
digestion was caused
by a decrease in Mg
2+
absorption and not by an increase in
endogenous Mg
2+
loss
(81)
.
Site of K
+
effect. A higher K
+
intake reduced Mg
2+
absorption
from the forestomachs. This reduction was not compensated for
in the small or the large intestine
(36)
. Furthermore, K
+
infusion
into the abomasum or ileum did not affect Mg
2+
absorption
(82)
.
The effect of K
+
and Mg
2+
concentrations. There is con-
siderable evidence showing that the effect of K
+
depends on
both ruminal K
+
and Mg
2+
concentration.
Role of K
+
intake: Inhibition of Mg
2+
absorption is pro-
nounced between 1 and 3 % K
+
in DM and is attenuated at
higher K
+
concentrations
(83)
. In agreement with this conclusion,
Schonewille et al.
(84)
did not nd a correlation between Mg
2+
digestion and high K
+
content of the diet within the range of 2·9
to 4·4 % of DM. Notably, Martens et al.
(58)
observed that the
absorption of Mg
2+
from the temporarily isolated rumen of
heifers dramatically decreased between 25 and 75 mmol K
+
/l in
the articial rumen uid, but not between 75 and 100 or 120
mmol K
+
/l. This agrees with the logarithmic relationship
between mucosal K
+
concentration and PD
a(48)
.
Role of Mg
2+
intake: The proposed model of job sharing
(Table 1) of the two uptake mechanisms suggests that the effect
of K
+
also depends on the Mg
2+
concentration. The reduction of
Mg
2+
absorption by K
+
must be higher if Mg
2+
is mainly trans-
ported via the K
+
-sensitive, PD-dependent mechanism. Ram
et al.
(64)
fed sheep increasing amounts Mg
2+
at two levels of K
+
intake. Mg
2+
absorption was reduced by 54 % at low Mg
2+
intake and by 27 % at high Mg
2+
intake.
The increase in K
+
intake elevates ruminal K
+(64)
and recipro-
cally decreases Na
+
concentration
(85)
. Neither the rumen volume
nor the passage rate was changed by K
+
intake, excluding dilution
of ruminal Mg
2+
concentration or enhanced outow
(64)
.
Meta-analysis of Mg
2+
digestion: reduction by K
+
The quantity of the effect of K
+
on Mg
2+
was analysed in a meta-
analysis by Weiss
(74)
in cows, yielding the following relationship:
Digestible Mg2+=45SEM 40ðÞg=d+024 SEM 007ðÞ
´Mg2+intake 44g=dðSEM 22Þ´K+;ð4Þ
where digestible Mg
2+
and Mg
2+
intake are given in g/d, and K
+
is
given as % K
+
in DM (thirty-nine diets, 162 cows).
Schonewille et al.
(24)
performed a second meta-analysis with
a different set of experiments and with a larger number of diets
and cows, yielding:
Mg2+true absorptionðÞ=36g=dSEM 067ðÞ+02SEM 001ðÞ
´Mg2+intake 008 g=dðSEM 0014Þ´K+;ð5Þ
where Mg
2+
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 Mg
2+
) by correction for endogenous Mg
2+
secre-
tion (700 kg BW ×4 mg/kg/d =2·8 g/d)
(86,87)
:
Mg2+apparent absorptionðÞ=36g=d28+02
´Mg2+intake 008 g=d´K+;ð6Þ
Digestible Mg2+=08g=d+02´Mg2+intake
008 g=d´K+;ð7Þ
Nutrition Research Reviews
Lumen Blood
1. Mg2+
2. Mg2+
Mg
2+
K
+
Mg
2+
K
+
P
C
Na
+
Mg
2+
C
Na
Na
+
2 A
Mg
2+
PD
a
PD
b
–+
PD
t
= PD
a
– PD
b
Mg
2+i
: 0·5–1·0 mmol/l
Imipramin
-
Subepithelial Mg2+ = 0·8–1·2 mmol/l
Ruminal Mg2+ = 2–10 mmol/l
Ouabain
-
-
DNP
ATP
ADP+P
pJ
ms
J
sm
Na
+
(K
+
, Ca
2+
)
-
+– +
+
+
Fig. 2. Representation of transepithelial ruminal Mg
2+
transport. The multi-layered
epithelium is simplified to one compartment. Passive Mg
2+
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
+
/Mg
2+
exchange via solute carrier family 41
member 1 (SLC41A1). The negative effects of inhibitors () on various steps of
Mg
2+
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; Mg
2+i
,
intracellular ionised (free) Mg
2+
; DNP, 2,4-dinitrophenol; A
, anion; C, carrier;
P, p u m p ( N a
+
/K
+
-ATPase). Example for PD
t
(+15 mV) =PD
a
(45 mV) PD
b
(60mV). Depolarisation of PD
a
by an increase of ruminal K
+
increases PD
t
.
Magnesium in ruminants 5
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Where Mg
2+
(apparent absorption), digestible Mg
2+
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 Mg
2+
digestion is slightly lower (20 %) than in the calculation of
Weiss
(74)
(24 %). However, Mg
2+
digestion is more depressed at
low Mg
2+
intake.
The linear decrease in digestible Mg
2+
with rising ruminal
K
+
(equations 4 and 7) is in contradiction to the discussed
reduction of an effect of K
+
at a higher Mg
2+
intake. The major
reason is probably the experimental design. The experiments
of Ram et al.
(64)
and Martens et al.
(58)
were performed
under identical conditions. Equations 4 and 5 are the result of
meta-analyses of many balance studies.
The role of Na
+
Insufcient Na
+
intake releases aldosterone and decreases Na
+
in both saliva and rumen uid, while K
+
is increased
(8890)
.
Accordingly, Na
+
deciency in sheep caused a decrease of
Na
+
in saliva and rumen uid, an increase of K
+
in both liquids,
and an enhanced PD
t
, while Mg
2+
absorption from the rumen
decreased (see Table 2)
(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
Mg
2+
concentration rose, while plasma Mg
2+
declined
(91)
. Since
aldosterone alone does not change Mg
2+
absorption
(92)
, these
effects are best explained by the aldosterone-induced elevation
of the ruminal K
+
concentration.
Notably, K
+
concentration in saliva can reach some
100 mmol/l in Na
+
-decient animals. Assuming a salivary ow
rate of 200 litres/d, this leads to a total inux of some 780 g K
+
/d
and presents a signicant risk for reduced Mg
2+
absorption. The
condition is easily overlooked, because overt clinical signs
of Na
+
deciency are usually missing and because the large
Na
+
pool in the rumen can be mobilised to cover deciency for
a long time
(93)
. Furthermore, as the K
+
concentration in the
rumen uid increases, the absorption of Na
+
is enhanced
(85)
,
which may help to compensate for Na
+
deciency.
Young spring grass frequently contains extremely low con-
centrations of Na
+(94)
and was suggested as a risk factor as early
as 1966 by Metson et al.
(95)
:If low sodium is conrmed 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
(96)
about a negative relationship
between the low Na
+
content of grass and the incidence of
tetany. Vice versa, grass tetany caused by Na
+
deciency can be
prevented by supplementation with NaCl
(97)
.
Protein and ammonia
Tetany-prone young grass in spring exhibits a high concentra-
tion of crude protein
(14)
, that causes an increase of up to
70 mmol/l ruminal ammonia
(98)
and is associated with grass
tetany
(99)
. (The term ammonia is used without discrimination
between NH
3
and NH
4
+
. Chemical symbols are used when a
specication is required.) Relationships between ammonia and
Mg
2+
absorption have been tested with contradictory results: both
inhibition of Mg
2+
absorption and no effect on Mg
2+
digestion at
high ruminal ammonia, depending on the experimental condi-
tions. A decrease in Mg
2+
absorption was observed at a sudden
increase in ruminal NH
4
+
concentration. Intraruminal application
of large amounts of ammonium acetate in cows caused a
decrease both in plasma Mg
2+
concentration and urinary Mg
2+
excretion
(76)
. When working with sheep
(34)
or young heifers
(58)
,
respectively, Mg
2+
absorption from the temporarily isolated
rumen was severely reduced by increasing NH
4
+
concentrations
which agrees with studies of the rumen pouch
(42)
.
However, alterations in Mg
2+
metabolism were not observed
in chronic experiments with a delay in sampling after raising
ruminal NH
4
+
concentrations
(100,101)
. These observations led to
the hypothesis that an acute increase in ruminal NH
4
+
reduces
Mg
2+
absorption, but that when ruminal NH
4
+
remains elevated
for a period of days, an adaptational response normalises Mg
2+
absorption. Gäbel & Martens
(101)
tested this hypothesis in vivo.
Acute addition of articial rumen uid with 40 mmol NH
4
+
/l into
the isolated sheep rumen signicantly reduced Mg
2+
absorp-
tion. In balance experiments, ruminal NH
4
+
was rapidly
increased from 4·81 (SD 0·18) to 47·9(SD 3·1) mmol/l within
1d.Mg
2+
excretion in urine transiently decreased from 385 to
255 mg/d over 2 d, but on the 3rd day, urinary Mg
2+
increased
and returned to control values, despite high ruminal NH
4+
(36·1(SD 4·8) mmol/l). Obviously, a sudden change in N intake
and NH
4
+
concentration impairs Mg
2+
absorption, but adapta-
tion occurred within 3 d.
The reason(s) for the temporary reduction of Mg
2+
absorption
by NH
4
+
have not been studied. Ammonia is transported across
the rumen epithelium both as NH
3
and NH
4
+
, depending on the
pH
(102)
. At a (physiological) pH of <7·0, NH
4
+
is predominantly
transported across cation channels in the apical membrane
(10,102)
,
decreasing PD
a
(103)
and increasing PD
t(101)
.Sinceavariable
fraction of the NH
4
+
that is taken up is extruded in the form of
NH
3
, protons are released and decrease the cytosolic pH
(10,103)
,
which stimulates apical Na
+
/H
+
exchange and Na
+
transport
(102)
.
These intraepithelial alterations of PD
a
and intracellular pH (pH
i
)
offer some suggestions. Firstly, PD
a
changes by some 10 mV
when NH
4
+
is elevated to 40 mmol/l
(103)
which may inhibit
channel-mediated uptake in analogy to what has been discussed
for K
+
. However, interactions between pH
i
and Mg
2+
transport are
a further possibility. Thus, the enhanced uptake of Na
+
due to
stimulation of Na
+
/H
+
exchange
(102)
should elevate cytosolic Na
+
(Na
+i
), which can be expected to interfere with basolateral
extrusion of Mg
2+
via Na
+
/Mg
2+
exchange. The possible
mechanisms of adaptation are still unclear.
Nutrition Research Reviews
Table 2. Na
+
deficiency and high K
+
intake change the same rumen
parameters and have identical effects on Mg
2+
absorption
Rumen
K
+
Na
+
PD
t
Mg
2+
absorption
High K
+
intake ↑↓ ↑
Na
+
deficiency ↑↓ ↑
PD
t
, transepithelial potential difference; , increase; , decrease.
6 H. Martens et al.
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Ruminal pH
Only unbound Mg
2+
in solution is available for transport across
the ruminal epithelium and, accordingly, chelating Mg
2+
by
EDTA strongly reduces Mg
2+
transport
(75)
. The range of free
Mg
2+
in the ruminal uid varies from 34 to 77 % of the total
amount
(104,105)
and depends on various factors
(78,105,106)
. One
major factor determining the digestion of Mg
2+
is the particle
size of MgO
(106)
. Furthermore, free Mg
2+
concentration in the
rumen depends on pH
(105)
. The curvilinear relationship
between rumen pH and Mg
2+
solubility exhibits a steep slope
between pH 5 and 7, which varies with diet
(105,107,108)
(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 Mg
2+
. An enhancing effect of low ruminal pH on
Mg
2+
digestion was suggested early on by Wilcox & Hoff
(109)
,
and is probably related to an increase in unbound Mg
2+
. Horn &
Smith
(107)
found a close and negative relationship between
rumen pH and Mg
2+
absorption before the duodenum. The
obvious effect of pH on ionised Mg
2+
is very likely the major
reason for the inuence of the diet on Mg
2+
absorption, parti-
cularly with regard to carbohydrates. A causal relationship
cannot be deduced from these studies, but the pH determines
Mg
2+
solubility with consequences for transport.
There is also reason to believe that Mg
2+
-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
(110)
. At present it is unclear if Mg
2+
conductance is
similarly affected. Conversely, an acidic pH has been shown to
decrease the expression of TRPM6 and other Mg
2+
-transporting
proteins
(111)
, which probably contributes to the renal Mg
2+
wasting that is observed in metabolic acidosis in man
(21,112)
.
Interestingly, chronic usage of proton pump inhibitors impairs
gastrointestinal Mg
2+
absorption
(113)
. The possible role of
ruminal pH in the aetiology of grass tetany is not clear, because
both higher
(107)
or lower pH has been reported
(114)
. However,
the close inverse correlation between ruminal pH and Mg
2+
absorption before the duodenum
(107)
suggests that a high
ruminal pH interferes with Mg
2+
digestion, particularly at low
DM intake in cold weather (H Meyer, personal communication)
or as a consequence of pre-existing subclinical hypomagne-
saemia with plasma Mg
2+
concentration 0·8 mmol/l and no
visible clinical signs such as ataxia or muscle spasms.
Mg
2+
absorption and readily fermentable carbohydrates
A low level of fermentable carbohydrates in tetany-prone grass
has been suggested to decrease Mg
2+
availability
(95)
. Vice versa,
drenching of grazing dairy cattle with a starch solution increased
plasma Mg
2+
concentration
(115)
and digestion of Mg
2+(57)
although
Mg
2+
absorption was not consistently improved
(116)
.Inruminal
uid, the addition of fermentable carbohydrates causes: (a) an
increase in the concentration of SCFA
(117)
, (b) a decrease in
pH
(117)
, which (c) enhances Mg
2+
solubility
(105)
, (d) a decrease in
NH
4
+
concentration, and (e) an increase of the number and size of
rumen papilla
(118)
, with the latter increasing the area for Mg
2+
absorption
(119)
.Hence,Mg
2+
digestion was enhanced in sheep by
lactose
(120)
.
The exact mechanism of the stimulation of Mg
2+
transport by
SCFA or HCO
3
/CO
2
is not clear
(60)
. Notably, addition of fer-
mentable carbohydrates to the diet with production of SCFA
enhanced Mg
2+
absorption from the caecum of rats
(121)
and, in
mice, inulin increased Mg
2+
absorption and expression of
TRPM6 and TRPM7 in the hindgut
(122)
. In studies with goats,
Schonewille et al.
(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. Inux of protonated SCFA with subsequent dis-
sociation can be expected to lead to cell swelling, which, in
turn, enhances monovalent currents both in cells hyper-
expressing TRPM7 channels
(124)
and in native ruminal epithelial
cells
(62,63)
. However, the most likely hypothesis is that the
PD-independent pathway is stimulated by SCFA. Replacement
of SCFA by gluconate signicantly reduced the J
ms
ux of
Mg
2+
and reduced uptake into cells
(59,61)
. This reduction in Mg
2+
transport does not reect binding by gluconate, because
gluconate only weakly binds Mg
2+(125)
, and does not affect the
epithelial transport of Mg
2+(126)
.
Mg
2+
intake and digestion
The meta-analyses of Weiss
(74)
and Schonewille et al.
(24)
demonstrated a linear correlation between Mg
2+
intake and
digestible Mg
2+
, suggesting a constant rate of Mg
2+
absorption
with no adaptation. However, McAleese et al.
(127)
orally dosed
28
Mg
2+
in sheep and observed a higher
28
Mg
2+
absorption at
decient Mg
2+
intake. In line with these ndings are the results of
Schweigel et al.
(52,56)
: incubation of isolated rumen epithelial cells
in a low- or high-Mg
2+
medium caused a corresponding increase
or decrease of in- and efux mechanisms of Mg
2+
. Although the
expression of TRPM7 was only slightly altered, both the expres-
sion of the Na
+
/Mg
2+
exchanger
(52,56,128)
, corresponding to
SLC41
(70)
, and the Mg
2+
channel MagT1 increased signicantly at
low Mg
2+
incubation and vice versa
(52)
, supporting the assump-
tion of the adaptation of Mg
2+
transport at low Mg
2+
.
Allsop & Rook
(129)
suggested that Mg
2+
absorption is sup-
pressed after increasing plasma Mg
2+
concentration by intra-
venous infusion and concluded that the most probable major
Nutrition Research Reviews
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14
Mg solubility (%)
Ruminal pH
Fig. 3. Scheme of Mg
2+
solubility in rumen fluid (redrawn from Dalley
et al.
(105)
). The slope of Mg
2+
solubility between pH 5 and 7 is influenced by
the diet (,).
Magnesium in ruminants 7
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site of action is therefore on the uptake of Mg from the reticu-
lorumen. Martens & Stössel
(23)
tested this hypothesis and
measured Mg
2+
absorption from the isolated rumen in sheep.
Mg
2+
(net) transport was not inuenced by increased plasma
Mg
2+
concentration or after 5 weeks of hypomagnesaemia,
which is in contrast to the suggestion of McAleese et al.
(127)
and
can probably be explained by the method used: sheep were
orally dosed with equal amounts of
28
Mg
2+
, and the appearance
of the isotope in blood was taken as Mg
2+
absorption. However,
it is highly likely that the ratio between the radioactive isotope
(
28
Mg
2+
) and the total concentration of Mg
2+
was much higher
in Mg
2+
-decient sheep than in controls. Accordingly, a higher
absorption of
28
Mg
2+
into blood could be expected even with-
out a change of the total rate of Mg
2+
a possibility that was not
considered by the authors since absorption from the rumen was
not known at that time.
Endogenous Mg
2+
secretion
Storry
(26)
made an estimation of Mg
2+
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 3·4and5·04 mg/kg found by Care
(130)
.
Asignicant part of the endogenous Mg
2+
loss is related to high
ow rates of saliva. In sheep, Dua & Care
(131)
estimated a secretion
of about 40 % of the Mg
2+
amount in the ECS or 23 mg/kg (live
weight). This involuntary endogenous loss of Mg
2+
is not constant.
In sheep on an articial, low-Mg
2+
diet, secretion dropped to
0·41·4mg/kg
(129)
, which is probably related to the linear correla-
tion between plasma Mg
2+
concentration and endogenous secretion
of Mg
2+
into the gut in general
(132)
, and into the small intestine
(31)
or
thebileinparticular
(130)
.
Schonewille & Beynen
(87)
summarised data for the endo-
genous Mg
2+
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 Mg
2+
absorption
The digestion of Mg
2+
in cows
(133)
and ruminal Mg
2+
transport are
inuenced by animal breed
(126)
. Greene et al.
(133)
have shown
that Mg
2+
absorption is greater in Brahman than in Jersey,
Holstein or Hereford cows. Leonhard-Marek et al.
(126)
measured
the net Mg
2+
transport in vitro across isolated rumen epithelium
of four breeds of sheep (Merino, Schwarzkopf, Skudde and
Heidschnucke). Skudde transported signicantly less Mg
2+
under
short-circuit conditions. The wide variation of Mg
2+
digestion seen
in different studies might have a genetic background and may
contribute to heritability of Mg
2+
in plasma
(16)
.Thesignicance of
a genetic variation of Mg
2+
transport proteins has been shown in
man, where mutation of TRPM6 channels reduced transcellular
Mg
2+
transport in the intestine and kidney
(50)
.
Vitamin D and Mg
2+
homeostasis
PTH and vitamin D
3
are the principal regulators of Ca
2+
meta-
bolism. Interactions between PTH, calcitriol and Mg
2+
in cows
are well established
(19,134,135)
, but the results are, in some cases,
contradictory
(136,137)
. Calcitriol increased plasma Ca
2+
and Mg
2+
concentrations in hypomagnesaemic sheep
(134)
and Ca
2+
con-
centration in cows, but decreased Mg
2+
concentration
(135,138)
.A
calcitriol-dependent uptake of Mg
2+
into soft tissue has been
suggested
(135,139)
. Calcitriol did not change faecal excretion in
cattle
(135)
, although calcitriol increased Mg
2+
absorption from
the rumen in sheep
(140)
.
The infusion of bovine PTH in cows caused an increase in
1,25-dihydroxyvitamin D
3
(1,25(OH)
2
D
3
), Ca
2+
and Mg
2+
in
plasma and a decrease in Mg
2+
in urine
(19)
, indicating enhanced
Mg
2+
resorption in the kidney. Dua et al.
(141)
observed a trend
for increased Mg
2+
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)
2
D
3
axis and
Mg
2+
metabolism can thus be observed, the physiological sig-
nicance of this interaction is not clear. The effect of 1,25(OH)
2
D
3
on epithelial Ca
2+
transport is classical and related to increased
expression and activity of TRPV5 and TRPV6 channels
(50)
.These
channels are non-selective cation channels with a high selectivity
for Ca
2+
over monovalent cations. However, a certain, albeit low,
permeability to Mg
2+
is to be expected. In summary, the possible
stimulation of Mg
2+
transport by 1,25(OH)
2
D
3
or effects of PTH
should be considered as a side-effect of Ca
2+
homeostasis.
Ionophores and Mg
2+
digestion
Ionophores like monensin and lasalocid signicantly increase
Mg
2+
digestion
(142)
. Both ionophores lowered ruminal K
+
con-
centrations in steers, suggesting a diminution of the reduction of
K
+
on Mg
2+
transport.
Sequestration of magnesium
A new environment, temperature changes or prolonged transport
of animals may lead to a shift in the distribution of Mg
2+
from the
ECS into the ICS
(143)
. The stress hormone adrenaline has
well-documented effects. Rayssiguier
(17)
intravenously infused
adrenaline in sheep and observed a rapid decline in plasma Mg
2+
concentration. This decrease was blocked by the β-receptor inhi-
bitor propranolol. Adrenaline or theophylline stimulates lipolysis
and increases NEFA, as a possible cause of sequestration
(144)
.Pre-
vention of both lipolysis and increase in NEFA by application of
sodium nicotinate abolished changes in plasma Mg
2+
concentration
in theophylline-treated sheep
(145)
.Furthermore,β-agonists such as
adrenaline activate the Mg
2+
channel TRPM7, stimulating uptake of
Mg
2+
into the cytosol
(146)
. Since TRPM7 is expressed throughout the
body, a sequestration of Mg
2+
into the cytosolic compartment is to
be expected. The pathogenesis of transport tetany probably
involves this adrenaline-dependent type of hypomagnesaemia
(147)
.
It may also play a role as a secondary factor in classical grass tetany,
in particular after the onset of the rst clinical signs and may
function as a trigger for tetanic muscle spasms.
Urinary Mg
2+
excretion
Adjusted renal handling (inux efux) is a precondition for
the regulation Mg
2+
homeostasis (Fig. 1) and includes two steps:
ltration and re-absorption
(21)
.
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8 H. Martens et al.
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Mg
2+
ltration
Plasma Mg
2+
varies from 0·9to1·2 mmol/l. Some 6080 % or
0·480·96 mmol/l of plasma Mg
2+
is ultraltrable so that, on a daily
basis, roughly 2959 g Mg
2+
/d will appear in the glomerular
ltrate of a cow of 650 kg BW (calculated with glomerular ltra-
tion rate (GFR) data of Murayama et al.
(148)
). Possible effects of
GFR on Mg
2+
ltration are not known.
Re-absorption proximal tubule. In the proximal tubule
2030 % of the ltered Mg
2+
is reabsorbed
(21)
. The fractional
reabsorption rate in this part of the nephron is remarkably con-
stant and probably occurs passively in an unregulated manner.
Re-absorption ascending limb of Henle. Most Mg
2+
(6070 %)
is reclaimed in the thick ascending limb of Henle (TAL)
(21)
.The
paracellular and passive transport in the TAL is mainly driven by
PD
t
(lumen positive). Energised by the basolateral Na
+
/K
+
-ATPase,
this potential is generated by the apical uptake of Na
+
,K
+
and
Cl
via NKCC2 with subsequent recycling of K
+
via renal outer
medullary K (ROMK) channels and basolateral extrusion via
ClC-Kb. Mg
2+
absorption is mediated by the tight junctional channel
protein, claudin-16 (paracellin-1), which interacts with claudin-19 to
form a cation-selective channel
(149,150)
. Reduction of the passive
driving force by blocking NKCC2 with furosemide
(151)
increases
magnesuria in sheep
(145)
.Mg
2+
transport in the TAL is stimulated by
PTH
(152)
in rabbits and, accordingly, a reduced urinary excretion of
Mg
2+
has been found after PTH infusion in vivo in cows
(19)
.
The passive transport across this pathway is regulated by Mg
2+
availability. Hypomagnesaemia in mice increases both claudin-16
protein and mRNA abundance, while Mg
2+
-loaded animals down-
regulated claudin-16
(153)
. The expression of claudin-16 is inhib-
ited by calcitriol
(154)
and further inuenced by a variety of
hormones such as glucagon, insulin, calcitonin, vasopressin or
isoproterenol
(155)
,whichmakesitdifcult to evaluate these effects
in vivo.Furthermore,Ca
2+
transport via claudin-16 is reduced by
Mg
2+(156)
:A competitive transport of Mg
2+
and Ca
2+
via the
common paracellular route in TAL could explain the coupling
between Mg
2+
and Ca
2+
excretion
(157)
(see below).
The remarkable roles of NKCC2, ROMK, CIC-Kb, and
claudins 16 and 19 in Mg
2+
homeostasis clearly emerge from
genetic studies in human subjects
(21)
.Thus,Mg
2+
homeostasis is
severely impaired by a mutation of the claudin-16 gene
(158)
.
Patients with this autosomal recessive disorder suffer from
hypomagnesaemia, hypermagnesuria and hypercalciuria. In
Japanese black cattle homozygous deletion (not mutation) of the
claudin-16 gene has been reported
(159,160)
, with reduced renal
Mg
2+
clearance and reabsorption
(161)
.
Re-absorption distal tubule. Approximately 510 % of the l-
tered Mg is reabsorbed in the distal convoluted tubule (DCT)
via active transport. Luminal Mg
2+
uptake is mediated by
TRPM6, driven by PD
a
(21)
. Renal TRPM6 is regulated by
epidermal growth factor, which has been considered to be the
rst autocrine/paracrine magnesiotropic hormone
(20)
.Mg
2+
decit increases TRPM6 mRNA and protein expression in
mice
(162,163)
. Neither PTH nor 1,25(OH)
2
D
3
stimulated TRPM6
expression in the kidney
(162)
. Interestingly, TRPM6 expression is
inuenced by the acidbase status of the animal. Metabolic
acidosis decreases renal TRPM6 expression and thus increases
Mg
2+
excretion, whereas metabolic alkalosis led to the opposite
effects
(111)
. The tight control of Mg
2+
transport by TRPM6 has
led to the conclusion that TRPM6 functions as a gatekeeper of
Mg
2+
. The efux mechanism across the basolateral membrane is
still uncertain, but may involve Na
+
/Mg
2+
exchange as in the
rumen
(12,128)
or the intestine (cyclin and CBS domain divalent
metal cation transport mediator 4; CNNM4)
(164)
.
The adaptation of Mg
2+
transport in the TAL and DCT has
raised questions regarding the signalling cascade. Particularly
intriguing is the rapid adaptation of Mg
2+
excretion by the re-
absorption of almost all ltered Mg
2+
under low dietary Mg
2+
intake, so that plasma Mg
2+
concentration is almost perfectly
maintained. Because mutation of the Ca-sensing receptor
(CaSR) causes disturbances of Mg
2+
homeostasis in man
(165)
, the
CaSR is emerging as an important player in the regulation of
reabsorption of both Ca
2+
and Mg
2+
via luminal and basolateral
sensing mechanisms
(21,157)
. More recently, Stuiver et al.
(166)
identied a protein (CNNM2), the mutation of which causes a
disturbance in Mg
2+
homeostasis. CNNM2 is located in the
basolateral membrane of the TAL and DCT, and is up-regulated
under Mg
2+
deciency. CNNM2 might contribute to a Mg
2+
sensing mechanism rather than transporting Mg
2+
itselfand
should thus considered to be a Mg
2+
homeostatic factor
(167)
.
Urinary Mg
2+
excretion
The adaptation of renal Mg
2+
transport activity in cows to various
levels of intake has been illustrated by Schonewille
(168)
and
Holtenius et al.
(169)
. Urinary excretion of Mg
2+
rises in a quasi-
exponential manner with plasma Mg
2+
concentration. However,
urinary Mg
2+
drops rapidly with falling plasma Mg
2+
, but levels off
at 0·610·73 mmol/l, after which Mg
2+
almost ceases to be
excreted in urine
(170)
. Accordingly, a dairy cow with a plasma
Mg
2+
concentration <0·8mmol/l has to be considered at risk of
hypomagnesaemia. This range of Mg
2+
concentration appears to
be a threshold. In a recent meta-analysis of Mg
2+
metabolism in
man, a concentration of 0·87mmol/l leads to substantial urinary
Mg
2+
excretion
(171)
.
Urinary Mg
2+
excretion is a more sensitive indicator of Mg
2+
availability than the plasma concentration. Rook & Balch
(172)
observed a much more pronounced decline of Mg
2+
in urine
than in plasma following a change in diet. The tight control of
Mg
2+
transport activity, particularly in the DCT
(21)
but also in the
TAL
(153)
, explains these classical observations.
The adjustment of renal Mg
2+
excretion to changes in dietary
intake with altered Mg
2+
absorption (inux) not only ensures
the maintenance of Mg
2+
homeostasis in most feeding situations
(Fig. 1), but also provides the practitioner with a diagnostic tool.
According to the data of Kemp
(173)
,Mg
2+
inux can be con-
sidered to be sufcient at urinary Mg
2+
>4·4 mmol/l, while a
range of 0·874·4 mmol/l might indicate a risk of Mg
2+
shortage.
Urinary Mg
2+
<1 mmol/l is probably a reliable indicator of
insufcient intake/absorption.
Interaction of magnesium and calcium
Mutual interactions of transport between Ca
2+
and Mg
2+
have
been observed
(174)
. Hypercalcaemia caused a large increase in
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Magnesium in ruminants 9
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urinary Mg
2+
excretion. Vice versa and again in rats, infusion of
Mg
2+
caused an increase of urinary Ca
2+
associated with a
reduction in Ca
2+
uptake via TRPV5
(175)
. A mutual interaction of
Ca
2+
and Mg
2+
has also been found in cows, with negative
interactions observed both on the level of the kidney
(176)
and
the rumen
(42,177)
.
Magnesium in milk
The Mg
2+
concentration in milk is much higher than in plasma
and exhibits a high heritability (0·60) in cows
(178)
. The higher
Mg
2+
concentration in milk requires active transport from
plasma to milk. Nothing is known about this mechanism, which
is most probably genetically determined and subject to modu-
lation or regulation, leading to the wide variation in milk Mg
2+
concentration. Cerbulis & Farrell
(179)
analysed Mg
2+
in the milk
of different breeds with a range of 99120 mg/l, with one cow at
268 mg/l. The average concentration of Mg
2+
in the milk
of all animals was 112 mg/l, close to the recommendation of
Schonewille & Beynen
(87)
of 120 mg/l. Assuming a milk yield of
3040 litres/d, a cow will lose some 35g Mg
2+
/d, which
approaches the total amount of Mg
2+
in the ECF (see Fig. 1). It is
important to realise that Mg
2+
efux via milk is continued
probably with some (genetic) variation even in hypomagne-
saemic cows
(180)
so that excretion of Mg
2+
in milk exacerbates
Mg
2+
deciency.
Goff & Horst
(181)
suggest that the concentration of Mg
2+
in
colostrum is 100 mg/l, although higher values of 238322 mg/l
were found by Shappel et al.
(182)
on the day of parturition in
heifers and cows, with a rapid and exponential decline post-
partum within 2 to 3 d to the normal level of 120 mg/l. The total
amount in colostrum on the day of parturition amounted to
1·574·97 g/d. The rapid change of Mg
2+
in milk after parturition
probably explains the much higher concentration of Mg
2+
in
early colostrum
(183)
. Kehoe et al.
(183)
reported 733 mg/kg
(range 2301399 mg/kg) in the colostrum of fty-ve fully
milked out cows from different herds within 4 h of calving.
Assuming a volume of 5 litres yields a rough estimate of 3·6g
Mg
2+
excretion in colostrum results, which underlines the
signicant Mg
2+
demand at parturition.
Magnesium and tetany
Plasma Mg
2+
and tetany
Sjollema
(13,14)
rst demonstrated the relationship between the
clinical symptoms of grass tetany and hypomagnesaemia.
However, the Mg
2+
concentration in the plasma of aficted
animals exhibits some variation (Table 3), and the severity of
the nervous disturbances is not closely related to the plasma
Mg
2+
concentration
(184)
. Possibly, the speed of plasma Mg
2+
decline promotes the onset of clinical manifestations
(185)
.
At values below 0·9 mmol/l, both an adequate supply of Mg
2+
or impending clinical hypomagnesaemia are possibilities, so that
a safe assessment of Mg
2+
status should involve a determination
of urinary Mg
2+
excretion. Even then, difculties in judging Mg
2+
status can be clearly seen in a study involving non-pregnant
lactating cows with normal Mg
2+
intake (2932·5g/d)and plasma
Mg
2+
concentration of 0·751·1 mmol/l
(186)
. After intravenous
infusion of Mg
2+
and despite a slight increase in plasma Mg
2+
in
four of the nine animals, the fractional renal Mg
2+
excretion
decreased, indicating Mg
2+
retention after the Mg
2+
load and
pointing towards a possible Mg
2+
decit. Despite these
uncertainties, low plasma Mg
2+
concentrations almost invariably
precede the onset of neurological symptoms with impaired
function of the CNS.
Clinical hypomagnesaemia. Classical hypomagnesaemic
tetany was originally observed a few days after cows had been let
out to graze in spring
(22)
.Atrst sight, it appears surprising that
the relatively large Mg
2+
pools in the ICS (130 g) or bones (about
320 g) of cattle cannot acutely be mobilised to maintain physio-
logical plasma Mg
2+(22)
, although a small mobilisation of 0·5g/d
has been reported in cows
(170)
, comparable with observations in
human subjects
(21)
. Mobilisation of Mg
2+
from bone is unlikely,
because the ratio between Ca
2+
and Mg
2+
in bone is 42 to 1, and
substantial withdrawal from bone would disrupt Ca
2+
homeo-
stasis
(187)
. Furthermore, both PTH secretion and sensitivity of
bone to PTH are decreased under conditions of hypomagnesae-
mia or alkalosis
(188)
. Cytosolic Mg
2+
is only partly available for
redistribution too; only 15 % is available in the ionised form with
the rest bound primarily to ATP or sequestered in microsomes
and mitochondria
(21)
. Accordingly, a massive efux of Mg
2+
from
the cytosol into the ECS might interfere with cellular energy
metabolism and cellular enzyme function.
Impaired function of the central nervous system. Hypo-
magnesaemic tetany is observed frequently as plasma Mg
2+
drops below 0·7 mmol/l
(189)
and was originally suggested to be
caused by impaired synaptic transmission at the motoric end-
plate
(190)
. This hypothesis was not conrmed by Todd &
Horvath
(191)
. The possible involvement of the CNS was rst
discussed by Chutkow & Meyers
(192)
at low Mg
2+
concentra-
tions in the cerebrospinal uid (CSF) of Mg
2+
-decient rats. The
hypothesis of a decreased Mg
2+
concentration in the CSF as a
reason for clinical signs was tested by Meyer & Scholz
(193)
in
Mg
2+
-decient sheep by measuring the Mg
2+
concentration in
plasma and CSF. They found that while the Mg
2+
concentration
in the CSF is kept constant over a wide range of plasma Mg
2+
concentrations, it begins to decrease at plasma levels <0·5
mmol/l so that at <0·25 mmol/l, Mg
2+
in CSF decreases almost
linearly with the concentration in plasma. Allsop & Pauli
(194)
further tested the discussed causal correlation between Mg
2+
in
CSF and clinical signs. Mg
2+
concentrations of <0·25 mmol/l in
the solution of CSF perfusion produced episodes of tetany that
were abolished by higher Mg
2+
concentrations. Because these
effects were not accompanied by changes in blood parameters,
Nutrition Research Reviews
Table 3. Status of Mg
2+
metabolism and plasma Mg
2+
concentration
Blood Mg
2+
Mg
2+
status mmol/l mg/100 ml
1. Normal Mg
2+
0·91·22·192·92
2. Uncertainty 0·80·91·952·19
3. Subclinical hypomagnesaemia 0·70·81·701·95
4. Symptomatic hypomagnesaemia <0·7<1·70
10 H. Martens et al.
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the clinical symptoms were considered to be caused by the
non-controlled activation of muscles by processes within the
CNS. However, little is known about the regulation of Mg
2+
in
the CSF. After rectal infusion of MgCl
2
, Reynolds et al.
(195)
observed that the Mg
2+
concentration in the CSF remained
constant in calves with normal plasma Mg
2+
, while in calves
with subnormal plasma Mg
2+
(<0·75 mmol/l), an increase in
Mg
2+
was observed in the CSF with a delay up to 120 min.
These results suggest carrier-mediated transport into the CSF
and might explain why a rapid decline of plasma Mg
2+
causes a
fall of Mg
2+
in the CSF, whereas a slow decrease allows for
sufcient Mg
2+
transport into the CSF. This conclusion agrees
with the observation of Allcroft & Burns
(185)
who suggested that
the speed at which plasma Mg
2+
level decreases is critical for
triggering clinical symptoms.
There are a number of reasons why a drop of Mg
2+
in the CSF
might trigger hyperexcitability. Mg
2+
is a physiological anta-
gonist of Ca
2+
-induced transmitter release at synapses
(196)
, and
low Mg
2+
in the CSF might facilitate Ca
2+
-dependent transmitter
release and the excitation of CNS neurons that, amongst others,
activate muscles. The activity of the glutamatergic NMDA
receptor (N-methyl-D-aspartate) in the CNS is inhibited by
external Mg
2+
in a PD-dependent manner and at low Mg
2+
in
the CSF, more receptors are activated, which should result in
hyperexcitability
(3,197)
. Furthermore, the activity of the inhibi-
tory γ-aminobutyric acid (GABA) receptor is enhanced by Mg
2+
.
Conversely, the inhibitory effects of GABA are reduced when
Mg
2+
falls, facilitating neuronal activation
(4)
. Hence, a decrease
of Mg
2+
in the CSF induces hyperexcitability of excitatory
neurons (NMDA) while reducing activity of inhibitory neurons
(GABA).
The effect of Ca
2+
concentration in the CSF on the onset of
clinical symptoms is still controversial. Reynolds et al.
(195)
and
Allsop & Pauli
(194)
observed diminished Mg
2+
and Ca
2+
con-
centrations in the CSF. However, plasma Ca
2+
concentration did
not correlate with clinical symptoms in sheep
(189)
.
Subclinical hypomagnesaemia
It is important to note that the appearance of clinically relevant
neurological symptoms is not obligatory, even when plasma
levels of Mg
2+
are low. Hypomagnesaemia of about 0·5 mmol/l
was induced in sheep by feeding a low-Mg
2+
diet for 5 weeks
without appearance of any neurological symptoms
(23)
. It is very
likely that Mg
2+
concentration in the CSF is maintained when
the induction of hypomagnesaemia with a low-Mg
2+
diet is
gradual (see above). It should be noted that even in the absence
of clear neurological symptoms, animals may suffer from var-
ious non-neurological manifestations of hypomagnesaemia.
Interactions between hypomagnesaemia and the regulation
of Ca
2+
metabolism were observed early on. Thus, Allen
et al.
(198)
showed a correlation between subclinical hypo-
magnesaemia 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
(136,199,200)
, the
functioning of PTH on the target organ
(201,202)
and the con-
version of 25(OH)D
3
to 1,25(OH)
2
D
3
(calcitriol)
(203)
. Moreover,
in organ cultures of fetal rat bone, the release of Ca by
supplementing 1,25(OH)
2
D
3
or PTH was reduced at low
(<0·8 mmol/l) Mg concentration
(204)
. Furthermore, regulation of
Ca homeostasis was found to deteriorate with induction of
secondary hypocalcaemia in calves with hypomagnesae-
mia
(200)
. These results correspond very well with in vivo
observations of Sansom et al.
(205)
, who found that the mobili-
sation of Ca from bone was lowered signicantly in cows with
hypomagnesaemia. A subsequent study by van de Braak
et al.
(206)
conrmed these ndings.
Conclusions and perspectives
A correlation between the clinical symptoms of grass staggers
or grass tetanyand hypomagnesaemia more than 80 years ago
initiated myriads of studies about Mg
2+
metabolism in rumi-
nants. These studies led to a stepwise improvement in under-
standing the pathogenesis: (a) hypomagnesaemia was not
caused by a Mg
2+
-decient diet, but by reduced availability
from the diet; (b) the site and mechanisms of Mg
2+
absorption
were described; and (c) the factors that inuence Mg
2+
transport
and digestion were characterised. Despite a considerable
increase in knowledge about the pathogenesis and prevention
of hypomagnesaemic tetany, many open questions remain.
Further work is necessary to identify if channels other than
TRPM6, TRPM7 or MagT1 contribute to PD-dependent uptake
of Mg
2+
in the rumen. In particular, the role of various other TRP
channels expressed by the rumen has to be claried
(10)
. The
PD-independent uptake mechanism is still not well char-
acterised and its molecular identity is unknown
(60,61)
. Further-
more, the antagonism between Mg
2+
and Ca
2+
in the gut and the
kidney deserves attention. In particular, renal excretion in vivo
is of major interest, because renal Mg
2+
transport is regulated
according to the Mg
2+
requirement. A better understanding of
this mechanism could lead to improved diagnosis of the Mg
2+
status of cattle.
Acknowledgements
The studies of the authors were supported by the Deutsche
Forschungsgemeinschaft (DFG). The manuscript was written by
H. M., and was improved and optimised by F. S., S. L.-M.
and M. R.
There is no conict of interest.
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