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Department of Nutrition, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands
2To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • magnesium absorption • wethers • potassium • potential difference
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), which
are common in areas with intensive livestock production (Fisher et al. 1994
). In controlled feeding trials with sheep and cows,
supplemental potassium bicarbonate decreases magnesium absorption
(House and Van Campen 1971
, Khorasani and Armstrong 1990
, Newton et al. 1972
,
Schonewille et al. 1999
). However, the inhibitory effect
of potassium was not observed in dry cows fed grass silages with high,
but variable potassium concentrations (Schonewille et al. 1997a
). The anion of potassium salts in grasses is
predominantly in the form of organic acids such as malate, citrate and
aconitate (Dijkshoorn 1973
, Grunes et al. 1992
, Marscher 1986
); thus, we hypothesized that
potassium intrinsically present in feedstuffs does not impair magnesium
absorption due to its anion. In a recent study, we rejected our
hypothesis (Schonewille et al. 1999
). However, there is
evidence to suggest that potassium chloride is less effective in
inhibiting magnesium absorption than is potassium bicarbonate
(Schonewille et al. 1997a
). To prove or disprove the
idea that the type of anion in potassium salts affects magnesium
absorption in ruminants, this study was conducted. Wethers were fed
equimolar amounts of potassium bicarbonate, potassium chloride or
potassium citrate, and apparent magnesium absorption was measured.
In ruminants, magnesium absorption takes place essentially in the rumen
(Rogers and Van't Klooster 1969
, Tomas and Potter 1976
) and consists of a potassium-independent,
carrier-mediated process and a potassium-dependent, electrogenic
transport component (Leonhard et al. 1989
). The amounts
of magnesium transported by the two mechanisms depend on the
concentration of magnesium in the soluble fraction of rumen contents
and thus on the ruminal pH, but the potassium-independent,
carrier-mediated process can become saturated with magnesium. Magnesium
absorption through the potassium-dependent, electrogenic transport
component is also determined by the transmural potential difference. We
investigated whether different potassium salts would inhibit magnesium
absorption to a different extent through different effects on the
determinants of the two absorption processes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experimental protocol was approved by the animal experiments committee of the Utrecht Faculty of Veterinary Medicine. Ruminally fistulated, 2-y-old wethers (n = 8), weighing 74 kg (SEM 0.84) were used. The wethers had been fistulated for at least 1 mo before the start of the experiment. The trial was conducted in the form of two 4 x 4 Latin squares with an interval of 2 wk. The four treatments consisted of a control diet low in potassium and three test diets enriched with one of three potassium salts. Within each Latin square, the wethers were randomly assigned to each sequence of feeding of the four experimental diets. Each Latin square was preceded by a 14-d preexperimental period. The experimental periods lasted 28 d. The wethers were weighed just before the evening meal on the last day of each dietary period. They were housed individually in pens with a layer of wood shavings or in metabolism cages with slatted floors.
Diets.
The experimental diets were formulated by the addition of equimolar
amounts of KCl, KHCO3 or K-citrate to the basal diet.
The ingredient composition of the pelleted concentrates (diameter
= 3 mm) and the total diets provided are shown in Table 1
. During the preexperimental period, all wethers were offered 219 g
pelleted straw, 219 g hay and 762.6 g low potassium concentrate.
The sheep were then fed one of the three experimental diets and the
preexperimental, control diet. The analyzed compostion of the
experimental diets is shown in Table 2
. The control diet contained 10.9 g K/kg dry matter, and the high K
diets contained 41.3 g K/kg dry matter, which is equivalent to 2.2
and 8.3 times the potassium requirement of adult sheep (NRC 1985
). The sheep were fed a restricted amount of feed to
maintain constant intakes of nonvariable nutrients. The diets were
offered three times per day in three equal portions at 0030, 0830 and
1630 h.
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The experimental feeds were sampled during each period, dried for 5 d at 60°C, ground and subsequently stored in a sealed jar at room temperature.
On d 17 of each experimental period, rumen liquid samples (~30 mL) were taken at 0900, 1100, 1300 and 1500 h. Immediately after collection, pH was recorded and the samples were centrifuged for 30 min at 30,000 x g. The supernatant was stored at -18°C.
From d 19 to 26 of each experimental period, urine and feces were collected quantitatively. The 24-h urine collections were weighed and 10% was stored at -18°C in a bottle that contained 50 mL of 6 mol/L HCl. The 24-h feces collections were stored at -18°C in plastic bags. At the end of each experimental period, the feces collections were pooled per wether and mixed thoroughly. Two samples, each representing 10% of total feces from each wether for each period, were dried for 5 d at 60°C, ground and stored in a sealed jar at room temperature (18oC). Similarly, the urine collections were pooled and two 100-mL samples were stored at -18°C.
Blood samples were taken on the last day of each experimental period, between 1500 and 1530 h. Blood was sampled from the jugular vein into evacuated heparinized tubes. The samples were centrifuged for 15 min at 2700 x g and the plasma was collected and stored in plastic tubes at -18°C.
Chemical analyses.
Nitrogen contents were determined by the macro-Kjeldahl method
(International Dairy Federation 1986
); a factor of 6.25
was used to convert grams of N into crude protein. Ether extracts of
the feedstuffs were prepared according to the Association of Official Analytical Chemists (1984)
; the solvent was evaporated
and the crude-fat residue weighed. The crude fiber contents of the
feedstuffs were estimated using the Fibertec System M2 (Tecator,
Stockholm, Sweden). Before the determination of the selected minerals
in feedstuffs and feces, the samples were ashed (480°C for 12 h)
and dissolved in 15 mL of 4 mol/L HCl. Magnesium, Ca and K were
estimated by atomic absorption spectroscopy and Na by atomic emission
spectroscopy (Perkin Elmer 3110, Perkin-Elmer, Norwalk, CT). Total
P in feedstuffs was determined by the method of Quinlan and DeSesa (1955)
. The accuracy of each assay run was monitored
using a commercial reference sample (hay powder, CRM 129, Community
Bureau of Reference, Brussels, Belgium) and in-house reference
samples; it was found to be within 5% deviation from the target
values. Magnesium in plasma and urine was measured directly by atomic
absorption spectroscopy. Sodium, K and Cl in the supernatants of
ultracentrifuged rumen liquid samples were estimated using an
ion-selective electrode (Beckman Instruments, Mijdrecht, The
Netherlands). Magnesium in the supernatants was estimated directly by
atomic absorption spectroscopy. The combined within- and
between-run precision of the determinations (CV) was 
3.0%
.
Potential difference.
The potential difference between rumen liquid and blood (jugular vein)
was measured as described by Dobson and Phillipson (1958)
. Measurements were done on the last day of each
experimental period for 7 h (0900–1600 h). The transmural
potential difference of four sheep was measured four times per second
using an analog-to-digital (A/D) converter (Data Acquisition Board, PCI 20428W-1, Intelligent Instrumentation,Tucson,AZ) built into a
computer. The A/D converter was connected with two separate reference
electrodes (Radiometer REF201, Copenhagen, Denmark), and each electrode
was dipped into a saturated KCl solution (3.5 mol/L). Saturated
KCl-agar bridges (1 g agar/20 mL saturated KCl solution) connected
the saturated KCl solutions to the sheep. The bridge to the rumen was
made by filling a polyethylene tube with saturated KCl-agar.
Contact with the rumen content was made by inserting the bridge through
the rumen fistula. The bridge to the blood was made by filling a nylon
tube with saturated KCl-agar and inserting it through a
polyethylene tube that was placed into the jugular vein. To prevent air
bubbles from being trapped at the tip of the catheter, ~1 L of saline
(154 mmol/L NaCl) was infused through the catheter during the 7 h.
Before statistical analysis, data for each animal were pooled for
periods of 1 h.
Statistical analyses.
All data were checked for normal distribution using the
Kolmogorov-Smirnov test (Wilkinson 1990
). Data were
not significantly different between the two Latin squares
(F-test); thus all data were pooled before statistical
analysis of treatment effects. It was reasoned that each day had three
identical postfeeding periods and that ruminal variables averaged for
the entire postfeeding period would be the correlates, if any, of
apparent magnesium absorption, i.e., magnesium intake minus fecal
excretion of magnesium. Thus, postfeeding ruminal variables were
averaged for each sheep and each period before statistical analysis.
All data were subjected to ANOVA with animal, experimental period and
dietary treatment as factors (Wilkinson 1990
). When the
influence of treatment was significant, Tukey's t test
was used to identify diets with different effects on the variable
involved. For the data from each sheep (n = 8) and
for each diet (n = 4), linear correlations were
calculated between ruminal variables and magnesium absorption. The
calculations were done under the assumption that the 32 data points
could be considered independently. To detect rumen variables that were
related to magnesium absorption, multiple regression analysis was
performed (Wilkinson 1990
) with sheep as factor,
magnesium absorption as dependent variable and rumen liquid
concentrations of magnesium and potassium, ruminal pH and transmural
potential difference as independent variables. Forward, stepwise
regression was performed by incorporating into the model the rumen
variable showing the highest significant, partial correlation
coefficient for its relation to the residual variance in magnesium
absorption. Throughout, the level of significance was preset at
P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The wethers consumed all feed supplied. Mean body weight, across all treatments, was 76 kg (SEM 0.79, n = 8). There was no significant influence of dietary treatment on body weight.
Magnesium absorption and plasma magnesium.
Intake of magnesium did not differ for all treatments (Table 3
). The addition of KHCO3 or K-citrate to the
diet significantly increased fecal magnesium excretion. Potassium
chloride did not significantly raise fecal magnesium excretion.
Consequently, apparent magnesium absorption was significantly decreased
after supplementation of the diet with either
KHCO3 or K-citrate, but not with KCl. The
decrease in magnesium absorption seen after feeding
KHCO3 or K-citrate was accompanied by a
significant decrease in urinary magnesium excretion. In contrast, the
diet with KCl did not affect urinary magnesium excretion.
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Minerals in rumen liquid and pH.
Magnesium concentrations in rumen liquid were not affected by dietary
treatment (Table 4
). Addition of potassium to the diet increased the potassium
concentrations in rumen liquid, irrespective of the type of salt. Rumen
concentrations of sodium and chloride were not significantly affected
by dietary treatment.
|
. Transmural potential difference.
The three diets supplemented with potassium raised the group mean
transmural potential difference (serosal side = positive), with
KCl producing the smallest rise (Table 4)
. The effects of
KHCO3 and K-citrate were significant, i.e.,
P = 0.004 and P = 0.001, respectively.
Correlations for individual values.
Apparent magnesium absorption did not correlate with rumen liquid
concentrations of magnesium (P = 0.491), potassium
(P = 0.311) or rumen liquid pH (P = 0.976), but there was tendency (P = 0.060) toward a
negative association with the potential difference across the ruminal
wall. The multiple regression model with a constant, the factor sheep
animal and rumen liquid concentrations of magnesium and potassium,
rumen liquid pH and transmural potential difference explained 31.5% of
the observed variance in magnesium absorption (P = 0.082). After forward, stepwise regression it appeared that only the
factor animal (P = 0.010) and the transmural potential
difference contributed significantly (P = 0.016) to the
explained variance in magnesium absorption (Table 5
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Khorasani and Armstrong 1990
,
Newton et al. 1972
, Schonewille et al. 1999
), showing that the addition of KHCO3
to the diet of ruminants lowers magnesium absorption. The
KHCO3-induced decrease in magnesium absorption
was 9.5%, which agrees with a previous experiment using wethers fed a
similar amount of KHCO3 (Ram et al. 1998
). On the basis of a comparison of different studies, we
predicted earlier that KHCO3 would reduce
magnesium absorption more effectively than an equimolar amount of KCl
(Schonewille et al. 1997a
). By direct comparison within
one study, we have now substantiated our prediction. The difference in
magnesium absorption for the diets containing either
KHCO3 or KCl reached significance only with
respect to urinary magnesium excretion, which can be considered an
index of magnesium absorption under maintenance conditions (Ram et al. 1998
, Schonewille et al. 1997b
). Compared
with the control diet , the intake of supplemental KCl did not
significantly reduce magnesium absorption. K-citrate, on the other
hand, significantly lowered magnesium absorption by 6.5%. This appears
to be the first feeding trial demonstrating that the lowering effect of
potassium on magnesium absorption is dependent on the type of anion in
the potassium salt ingested. Even though magnesium absorption was
depressed by supplemental KHCO3 and
K-citrate, hypomagnesemia did not occur. Thus, in this study, the
wethers absorbed sufficient magnesium to maintain magnesium balance as
indicated by Table 3
.
In vitro studies with rumen epithelium (Leonhard-Marek and Martens 1996
) have indicated that the inhibitory action of
potassium on magnesium absorption can be explained by the depolarizing
action of potassium on the apical membrane potential of rumen
epithelial cells, which reduces the driving force for magnesium uptake
by these cells. The reduced apical membrane potential difference
results in an increased transmural potential difference associated with
depressed magnesium absorption (Martens and Blume 1986
,
Martens et al. 1987
). This feeding trial supports the
mechanism proposed on the basis of studies with isolated rumen models.
The feeding of KHCO3 significantly elevated the
transmural potential difference. K-citrate administration also
significantly lowered magnesium absorption associated with a marked
increase in the potential difference. The elevating effect of KCl on
the potential difference was intermediate as was its lowering influence
on the percentage of magnesium absorption. Multiple regression analysis
showed that there was a significant, negative correlation between the
potential difference and the percentage of magnesium absorption (Table 5)
.
It can be concluded that the different effects of
KHCO3 and KCl on magnesium absorption are
secondary to the different effects of these salts on the potential
difference. The different effects of the salts were unrelated to the
potassium concentration in the rumen fluid (Table 4)
, supporting the
theory that the type of anion affects the potential difference. Indeed,
the potassium concentration in rumen liquid alone was weakly associated
with the potential difference across the ruminal wall (r
= 0.323, n = 32, P = 0.093), but
when the chloride concentration in rumen liquid was added to the model,
both potassium and chloride contributed significantly (P
= 0.015 and P = 0.026, respectively) to the
explained variance in the transmural potential difference
(R2adj = 21%,
P = 0.020). Furthermore, in vitro studies have shown
that chloride vs. sulfate lowers the potential difference
(Ferreira et al. 1966
, Martens and Blume 1986
). Thus, potassium and chloride appear to have opposite
effects on the transmural potential difference.
In addition to diffusion, ruminal magnesium absorption also involves a
carrier-mediated process that is based on exchanging a magnesium
ion for two hydrogen ions (Martens et al. 1991
,
Scharrer and Lutz 1990
). The two transport mechanisms
are dependent on the magnesium concentration in the rumen fluid, even
though the carrier-mediated mechanism can become saturated
(Brown et al. 1978
, Care et al. 1984
,
Martens and Harmeyer 1978
, Martens 1979
).
Thus, it could be suggested that dietary potassium also reduces
magnesium absorption by lowering the concentration of magnesium in the
rumen liquid. The soluble-magnesium concentration, in turn, depends
on the ruminal pH. At pH values >6.0, there is a sharp fall in the
concentration of soluble magnesium (Dalley et al. 1997
).
There was no relation between either ruminal pH or magnesium
concentration and magnesium absorption. The data in Table 4
indicate
that only KHCO3 feeding raised the ruminal pH and lowered
magnesium concentrations. K-citrate also raised the group mean
ruminal pH but did not lower magnesium concentrations. It seems that a
pH-induced change in magnesium concentration was not an important
determinant of magnesium absorption in our wethers fed different
potassium salts.
In conclusion, this study shows that the type of anion in potassium salts has an effect on magnesium absorption in addition to the inhibitory effect of potassium itself. Compared with the control diet, supplemental KHCO3 and K-citrate significantly lowered the percentage of magnesium absorption, whereas KCl did not. On the basis of this study, the salt effect relates to the transmural potential difference. Compared with the control diet, supplemental KHCO3 and K-citrate significantly raised the potential difference, whereas KCl did not.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received May 6, 1999. Initial review completed June 4, 1999. Revision accepted July 19, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Martens H., Blume I. Effect of intraruminal sodium and potassium concentrations and of the transmural potential difference on magnesium absorption from the temporarily isolated rumen of sheep. Q. J. Exp. Physiol. 1986;71:409-415
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