Effects of Reducing on Calcium Kinetics Dietary Cation-Anion Balance in Sheep‘ ABSTRACT A Ca kinetic study with a four- compartment model beiig fitted to radi- oisotope and balance data using the CONSAM (conversational, simulation, analysis, and modeling) computer pre gram was conducted to examine the ef- fects of dietary cation-anion balance, cal- culated as milliequivalents [(Na + K] - (Cl + S)]. Twelve crossbred wethers were used as eucalcemic control (period 1); then Ca loss during lactation was simulated by continuous infusion of ethylene glycol tetraacetate (period 2). Dietary cation-anion balance was man- ipulated by supplementation of various mineral salts and was +339, +35, and -127 meq of kg DM-’ during period 1 and d29, +68, and -147 meq of kg DM-l during period 2 for control and two treatments, respectively. Animals re- sponded to the simulated lactational Ca loss (period 2) by inmasing true inesti- nal absorption of Ca and bone resorption and by reducing Ca accretion by bone. No difference was observed in concen- tration of total Ca in plasma, but treat- ments produced increased concentration of plasma ionized Ca during both peri- ods. Both treatments produced hypeml- ciuria during both periods, and the lowest cation-anion balance increased true intestinal absorption of Ca and reduced bone accretion during period 2. The size of total exchangeable Ca pool Received April 30,1990. ''Ibis research was partly sujprted by grant G1443 of the Natural Scieoces aod Engineering Research Cooocil of Canada and by the Ministhe de I’Enseigmment Supkrieur de la Science et de la Teclmologie du Qotbec. Accepted May 2,1991. 2~~~ to wimm to address reprint regoests. HlROSHl TAKA01 and ELLIOT BLOCK2 Department of Animal Science Macdonald College of McGill University Ste. Anne De Bellevue, Po, Canada H9X 1CO did not differ between treatments or peri- ods, but amount of Ca movement be- tween the pools increased with the inter- mediate cation-anion balance during period 1 and with both treatments during period 2 compared with controt These results indicated that feeding reduced cation-anion balance diets increased Ca flux through the exchangeable Ca pool with no changes in the size of the pool, particularly when Ca demand was in- CreaSed. (Key words: cation-anion balance, min- eral metabolism, calcium kinetics, sheep) Abbreviation key: DCAB = dietary cation- anion balance, EGTA = ethylene glycol-bis (p-amino-ethyl ether) N,N,”,N’-tetraacetic acid, PTH = parathyroid hormone, SA = spe- cific activity, TRT = treatment. INTRODUCTION The Ca homeostatic mechanism operates very tightly to maintain extracellular Ca within physiological ranges (8 to 11 mg d-l). A change in physiological status, such as the hitiation of lactation, creates a rapid distur- bance in this mechanism. Plasma Ca ex- changes with a large mass of Ca in soft tissues and bone surfaces, which may function to buffer the effect of such rapid changes in these pools. The size of the exchangeable Ca p l can be determined from kinetic analysis of plasma-specific activity of Ca after administra- tion of intravenous radioactive tracer (3). Dietary cation-anion balance @CAB), de- fined as milliequivalents of (Na + K) - (Cl + S), can cause changes in Ca homeostasis. A negative DCAB has excessive amounts of an- ions in relation to cations in the diet and is considered acidogenic in nature. It was sug- gested that acid-base balance as affected by feeding acidogenic diets alters Ca metabolism 1991 J Dairy Sci 7442254237 4225
Transcript
Effects of Reducing on Calcium Kinetics
Dietary Cation-Anion Balance in Sheep‘
ABSTRACT
A Ca kinetic study with a four- compartment model beiig fitted to radi- oisotope and balance data using the CONSAM (conversational, simulation, analysis, and modeling) computer pre gram was conducted to examine the ef- fects of dietary cation-anion balance, cal- culated as milliequivalents [(Na + K] - (Cl + S)]. Twelve crossbred wethers were used as eucalcemic control (period 1); then Ca loss during lactation was simulated by continuous infusion of ethylene glycol tetraacetate (period 2). Dietary cation-anion balance was man- ipulated by supplementation of various mineral salts and was +339, +35, and -127 meq of kg DM-’ during period 1 and d29, +68, and -147 meq of kg DM-l during period 2 for control and two treatments, respectively. Animals re- sponded to the simulated lactational Ca loss (period 2) by inmasing true inesti- nal absorption of Ca and bone resorption and by reducing Ca accretion by bone. No difference was observed in concen- tration of total Ca in plasma, but treat- ments produced increased concentration of plasma ionized Ca during both peri- ods. Both treatments produced hypeml- ciuria during both periods, and the lowest cation-anion balance increased true intestinal absorption of Ca and reduced bone accretion during period 2. The size of total exchangeable Ca pool
Received April 30,1990.
''Ibis research was partly sujprted by grant G1443 of the Natural Scieoces aod Engineering Research Cooocil of Canada and by the Ministhe de I’Enseigmment Supkrieur de la Science et de la Teclmologie du Qotbec.
Accepted May 2,1991.
2~~~ to wimm to address reprint regoests.
HlROSHl TAKA01 and ELLIOT BLOCK2 Department of Animal Science
Macdonald College of McGill University Ste. Anne De Bellevue, Po, Canada H9X 1CO
did not differ between treatments or peri- ods, but amount of Ca movement be- tween the pools increased with the inter- mediate cation-anion balance during period 1 and with both treatments during period 2 compared with controt These results indicated that feeding reduced cation-anion balance diets increased Ca flux through the exchangeable Ca pool with no changes in the size of the pool, particularly when Ca demand was in- CreaSed. (Key words: cation-anion balance, min- eral metabolism, calcium kinetics, sheep)
The Ca homeostatic mechanism operates very tightly to maintain extracellular Ca within physiological ranges (8 to 11 mg d-l). A change in physiological status, such as the hitiation of lactation, creates a rapid distur- bance in this mechanism. Plasma Ca ex- changes with a large mass of Ca in soft tissues and bone surfaces, which may function to buffer the effect of such rapid changes in these pools. The size of the exchangeable Ca p l can be determined from kinetic analysis of plasma-specific activity of Ca after administra- tion of intravenous radioactive tracer (3).
Dietary cation-anion balance @CAB), de- fined as milliequivalents of (Na + K) - (Cl + S), can cause changes in Ca homeostasis. A negative DCAB has excessive amounts of an- ions in relation to cations in the diet and is considered acidogenic in nature. It was sug- gested that acid-base balance as affected by feeding acidogenic diets alters Ca metabolism
1991 J Dairy Sci 7442254237 4225
4226 TAKAGI AND BLOCK
(23, 27, 29, 32) via bone resorption (31, 33, 38). intestinal Ca absorption (7, 49), and renal handing of Ca (5, 43, 45). Because of effects of DCAB on Ca metabolism, several studies have been conducted, these studies have demonstrated that feeding reduced or negative DCAB (acidogenic) to prepartum dairy cows decreased the incidence of peripamuient hypo- calcemia (milk fever), whereas feeding posi- tive DCAB (alkalogenic) increases its inci- dence (7, 18, 19, 20). However, effects of manipulating DCAB on Ca kinetics have been reported only in one study (25) with goats.
The objective of this experiment was to determine the effect of changing DCAE3 (achieved by supplementation of various min- eral salts) on Ca kinetics using a four compart- ment model in sheep during a period of normal Ca status (eucalcemic) and during simulated lactational Ca losses caused by continllous in- fusion of a 4.5% solution of ethylene glycol- bis (~-amin&yl ether) N,N,","-tetraa~etic acid (EGTA).
MATERIALS AND METHODS
Animals and Diets
The experiment was conducted with 12 wethers (Suffolk crossbred) at an average age of 2 yr. Animals were placed in individual metabolic cages in a room with temperature maintained at 2o'C and with a photoperiod of 14 h of light:10 h of darkness. Prior to com- mencing the experiment, animals were treated
TABLE 1. Ration composition for sheep fed different cation-anion balanced diets (DCAEI).'f
'DCAB (maq kg-' of DM): = 384, TRT A = 52; TRT B = -137.
for gastrointestinal parasites with Levamisol (Cyanamid Canada, Montdal, PQ, Can.).
Three DCAB were formulated by adding various mineral salts to a basal diet composed of corn silage and chopped alfalfa hay; posi- tive control, near zero treatment (TRT) A, and negative TRT B. Composition of diets and mind premixes ate presented in Tables 1 and 2, respectively. Mineral premixes were formu- lated to meet trace mineral requirements of animals (36) and to deliver excess anions or cations, depending on d i w treatments. All mineral salts utilized were of laboratory grade. Rations were offered as TMR twice daily at 0830 and 1600 h. Feed and orts samples were taken daily at the moming feeding and frozen for analysis.
Twelve lambs were assigned to four blocks according to their BW, within each block, they were randomly assigned to one of three diets in a random block design. Each study of 35 d consisted of an initial adjustment period of 14 d and two collection periods of 7 d each with a 7d interval between them. From d 8 of the
TABU 2. Composition of mineral mixtures for diets with varying cation-mion CAB).'*^^*^ Minaal salt CTR TRTA TRTB
b i n d mixtures p v i d c the following; 20 ppm of Mu, 5Oppmof Fe, 6 ppmof Cu, .8 ppmof Co, .6 ppm of
%itamin A (71250 IU kg-' of mix) and vitamin D
TRT B = -137.
I, 75 ppm of zn.
(26250 IU k& of mix) wae added
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CALCIUM KINETICS AND CATION-ANION BALANCE 4227
adjustment period and thereafter, animals within each block were pair-fed to the level of lowest consumption within the block in order to equalize feed intake between dietary treat- ments. On d 14 of the adjustment period, animals were restrained, and both jugular veins were catheterized. The initial collection period of 7 d was designed to establish effects of experimental diets on Ca kinetics during the eucdcemic state. Radioactive Ca as 4 % 2 a 2 in aqueous solution with a specific activity of 10.3 to 12.3 Ci g1 was adjusted to contain 80 pCi ml-'. An injection of 5 pCi kg-' was administered via one of the jugular vein catheters at O900 h on d 1 of the collection period. The second collection period was iden- tical to the first, except that, at 24 h prior to injection of 45Ca and thereafter, EGTA (Sig- ma, St. Louis, MO) was continuously infused intravenously at a rate of 55 mmol d-l, thereby producing a standardized rate of Ca loss. A 1:l chelation ratio of EGTA to Ca was assumed (22), and the amount of EGTA infused was calculated to simulate a lactational Ca loss with a milk production of 1.3 kg of milk d-' for sheep, assuming the content of Ca in milk at 1.7%. This Ca loss is proportional to that of a dairy cow at the initiation of lactation.
The pH of the EGTA solution was adjusted to 7.4 with 5N NaOH the antibacterial agent, Trivetrin (Coopers, Willowdale, ON, Can.), also was added (5 ml L-l). This solution was delivered by peristaltic pump (Minipuls 2, Level, France) with an approximate infusion rate of 19.5 ml t-'. To ensure sterility of the solution, the infusate was passed through a syringe filter (.45 pm, Nalgene Co., Rochester, NY).
Details of housing and feed, orts, and urine and feces collection have been described else- where (46, 47). During the collection period, additional daily samples of urine and feces were obtained for radioactivity measurements prior to the morning feeding.
Blood sampling was carried out in an ex- ponential fashion after 45Ca injection: every 30 s from 0 to 5 min postinjection, each 1 min from 5 to 10 min, every 2 min from 10 to 20 min, every 5 min from 20 to 30 min, every 15 min from 30 to 150 min, and every hour from 4 to 11 h postinjection. Additional samples were collected from d 2 to 7 of the collection period every 3 h between O900 and 2100 h.
Blood samples were transferred immediately to 10-ml test tubes kept on ice and centrifuged at 750 x g for 15 min to collect plasma within 2 h of sampling.
Analyses
Feed, orts, and feces samples were placed in a ford-air oven for 72 h for DM determina- tion and were ground through a 2-mm screen in a hammer mill. For mineral determination, feed, orts, and feces were first wet ashed with a mixture of concentrated nitric and perchloric acids (101) for 6 h. The digested solution was analyzed for Ca, Mg, Na, K, Fe, and Al by a Perkin-Elmer 2830 atomic absorption spectro- photometer (Perkin-Elmer, Norwall, cr) at a g propriate dilution rates.
The determination of inorganic S content in feed, orts, feces, urine, and plasma were described by Takagi and Block (46). For the measurements of C1, a modified method of potentiometric titration procedure (34) was employed using a Cl-specific ion electrode. Feed and orts (.5 g) and feces (2 g) were suspended in 20 ml of .lN nitric acid and vigorously mixed at 10- to 15-min intervals for at least 1 h; concentration of C1 in the sus- pended solution was determined by a double end point titration method with .0282N AgNO3 as the titrant.
Plasma mineral determinations have been described by Takagi and Block (47). Ionic Ca in plasma was determined by Nova 7 (Nova Biomedical, Waltham, MA), and values were normalized to plasma pH of 7.40.
Ground feces samples (1 g) were ashed overnight at W C in a furnace (Thermolyne Sybron, Dubuque, IA), and the ash was dis- solved in 2Wo vol/vol HCl, evaporated to dry- ness on a hot plate at l W C , then redissolved in 20% vol/vol HCl, and brought up to a volume of 10 ml with 20% vol/vol HCI. After being centrifuged at 500 x g for 15 min, the clear Supernatant was collected. One milliliter of the Supernatant and UnaciWied urine (1 ml) or plasma (1 ml) were dissolved with 9 ml of scintillation cocktail (Universal cocktail, IC" Biomedical Inc., Montr6al, PQ, Can.) in a 20-ml scintillation vial, and radioactivity was measured by liquid scintillation spectrometry (1209 Rackbeta, LKB, Bromma, Sweden). Quenching curves for plasma, urine, and feces
Journal of Dairy Science Vol. 74, No. 12, 1991
4228 TAKAGI AND BLOCK
were constructed separately by mixing varied amounts of normal, nonradioactive samples with known amounts of 4 5 ~ a utilized to obtain disintegrations per minute of samples. Specific activity (SA) of the plasma then was expressed as a fraction of the injected dose per gram of Ca; excretion of radioactivity in urine and feces was expressed as a cumulative fraction of injected dose.
Kinetic Analysis
The four-compartment series model of Ca kinetics of Ramberg et al. (40) was adapted for ruminants from a model of normal humans (37). The model consists of a collection of interconnected compartments in a steady state (equilibrium). Four compartments represent the number of exponents required to describe best the plasma SA decay curve over a period of 7 d (2) and is described by the equation
where
R,(t) = plasma SA at time t, A, to = “intercept” terms with dimen-
sion of concentration of rate constants, and
a1 to a4 = the overall distribution and elimination rate constants.
The four individual compartments represent regions or homogenous chemical spaces that have identical transition probabilities (turnover rate) of exchangeable Ca (2) rather than spa- rate physical entities @e., organ). The nota- tions follow those of Aubert and Michaud (3) and Aubert et al. (2):
M = unit of mass between which various Ca exchanges occur, and
V = the rate in units of mass per unit time of an exchange process.
Notation for unidirectional processes and for exchange (in grams per day) is as follows:
Vi = dietary Ca intake, VF = total fecal Ca excretion,
B = Ca balance, Vf = endogenous fecal Ca excretion, V,, = urinary Ca excretion, V, = Vi + Vf - VF = the amount of dietary
Ca that was absorbed, VOt = unidirectional movement of Ca from
the system into bone (accretion), V& = movement of unlabeled Ca from bone
into the system (resorption), Mi = the mass of Ca in compartment i,
Rij = rate of stable Ca transport from j to i
Compartments 1 through 4 represent dilu- tion of isotope in progressively larger masses of Ca that together constitute the total ex- changeable Ca pool (MT), Compartment 1 is the site of tracer injection and is sampled via the blood. It was assumed that inflow of Ca from the intestine (VJ and from bone (V,) enter compartment 1 and that excretory losses of Ca to feces (Vf) and urine (V,,) leave only from compartment 1, whereas compartment 4 is the site of loss of Ca to nonexchanging bone
The computer program, CONSAM [conversational, simulation, analysis, and modeling (5)] , was used to fit the model for each data set. All sample radioactivities (counts per minute) were adjusted for decay, background, and quenching; plasma samples were expressed as SA (dpm g-’ of Ca) as a proportion of injected dose. Cumulative urine and feces radioactivity (dpm) also were ex- pressed as a proportion of injected dose.
Steady state parameters were determined after estimates were entered for proportionality (K,) and rate (L,j) constants. Then, the pro- gram adjusted those initial estimates of the parameters by an iterative process until a least squares fit of the data was obtained. A best fit solution was obtained when the residual sums of squares reached a minimum value. The model type used was linear differential equa- tions with constant efficiencies (16). The final solution included the parameter values &j), estimate of their uncertainty, and the steady
MT = Mi + M 2 + M 3 + %, and
(Vo+).
state solution (Mi and Rij).
Statistical Analysis
Dietary treatment effects on mineral bal- ance, blood traits, and 45Ca kinetic expressions
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CALCIUM KlNETICS AND CATION-ANION BALANCE 4229
TABLE 3. Mined composition of three diets fed to sheep duriog two physiological states (eucalcemic and EGTA infusion).'
PPm 411 405 1278 37 1 257 1454 4 P P m 359 1164 856 114 1075 1128 Cation-anion balance? mea kz-l of DM 338 35 -127 429 68 -147
1cr~ = control; TRT = tmmmq EGTA = ethylem glycol tetraacetate. %he E T A infusion used io simulate lactational Ca loss in blood. 3~alcalated as milliequivdents of ma+ + K+) - (a- + ST kg-' of DM.
were evaluated by ANOVA for a random block design, and Scheffe's test (44) was uti- lized for comparative purposes between the treatments. Comparison between eucalcemic and EGTA-infused sheep was evaluated by r test for paired observations (44). All statistical analyses were carried out using SAS (SAS Institute, Inc., C q , NC). Significance of 10% was used throughout unless otherwise noted. The reader should be aware that infusion and period are confounded effects, because the EGTA infusion always followed a eucalcemic period. Therefore, the possibility exists that effects between infusions are due to period. However, the authors assume that, because blocb were assigned to treatments at different times of the year and effects were consistent between blocks (Le., no effect of block), any confounding effects are minimal, if they exist at all. Statistics within an infusion period are not confounded.
RESULTS
Mineral composition of diets is shown in Table 3. Dietary Ca (.88 to 1.06% DM) and Ca to P ratio (2.93 to 3.39) were above reQuire ments [.42% and 2.0, respectively (36)]. Cation-anion balance of diets for control, TRT A, and TRT B, respectively were +339, +35, and -127 during the eucalcemic period (period 1) and +429,+68, and -147 during the EGTA infusion period (period Z), respectively.
Body weight, feed intake, feces and urine volume, and daily DCAB are presented in Table 4. No differences were observed in feed intake because of the pair-feeding regimen. Urine volume was increased (P < .05) by EGTA infusion. Intakes of DCAB were +375, +37, and -136 meq ti1 in period 1 and +493, +79, and -210 meq d-lin period 2 for control, TRT A, and TRT B, respectively.
The infused volume of EGTA varied be- tween 456 to 476.2 ml d-l, and the loss of Ca from the exchangeable Ca pool was calculated to be appmximately 55 mmol d-l, which was equivalent to a milk production by sheep of 1.3 kg d-I (assuming Ca concentration in milk of 1.7%).
Concentrations of plasma minerals are shown in Table 5. The mean total concentra- tion of Ca in plasma was calculated using more than 70 samples collected over the 7 6 collection period. Concentration of plasma Ca did not differ between treatments; however, EGTA infusion increased (P < .Ol) the concen- tration of total plasma Ca. The ionic form of plasma Ca (Ca*) was affected by treatments; sheep fed reduced or negative DCAB (TRT A and TRT B) had higher Ca* (P c .01) than control during both periods, and this was reflected in the ratio of Ca* to total Ca.
Concentration of plasma inorganic P was the only other mineral (Table 5) affected by dietary treatments. Animals fed control had higher (P c .01) values than for TRT B during
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4230 TAKAGI AND BLOCK
TABLE 4. Body weight, feed intake, excreta outpu& and daily intake of cation-anion balance by sheep fed d ~ e r e n t cation-anion balanced diets (DCAB) during the eucalcemic and EGTA infosion periods.'*2
Pcriod Cl'R TRTA TRTB SEM Cla T R T A T R T B SEM effect
1.75 .13 urine excretion, L K' 1.17 1.10 1.35 .13 1.46 1.54 * Intake of anion-cation balance, meq d-' 375.7' 37.2b -136.2' 40.7 493.5' 78.6b -209.p 13.2 NS
"b7cMeans with different superscripts within period differ (P < .05). 'CTR = control; TRT = heatment; EGTA = ethylene glycol tetraacetate. %he DCAB for CIR, TRT A, and TRT B were 338, 35, and -127 meq kg-' of DM in eucdcemin period and 429,
*P c .05. 68, and -147 rneq kg-' of DM in EGTA infusion period.
the eucalcemia period, and sheep fed control had higher (P e .01) values than for both TRT A and TRT B during the EGTA infusion peri- od. In addition, plasma P was positively corre- lated with intake of DCAE (r = -52, P = .009).
Sheep fed both TRT A and TRT B sharply increased urinary excretion of isotope com- pared with control during the eucalcemic pe- riod (Figure l). However, this difference be- came smaller during the EGTA infusion period, because urinary isotope excretion dur- ing this period represented a combination of
endogenous urinary Ca and EGTA-chelated Ca, which represents the simulated lactational Ca loss. Total isotope excretion (urine and feces) by animals fed the control during the eucalcemic and EGTA infusion periods was comparable with that of nonlactating and lac- tating cows, respectively (40).
The early portions of the cumulative fecal excretion curves (first 2 d) showed a delay in the appearance of isotope excrete therefore, only the latter portion (d 3 to 7) of the data points was utilized in the process of fitting the
TABLE 5 . Blood parameters of sheep fed merent cation-anion balanced diets during the eucalcemic and EGTA infusion periods.'
E u c a l d c period CTR TRTA TRTB SEM
Toral Ca, mg dt-' 8.81
Ionic/total Ca, % 38.8b
N4 meq L-' 150.4
Cl, maq L-l 109.5
Ionic ca, mg dr' 3.3b
P, mg d-' 4.7a
K, m q L - ~ 4.9
s. mmol L-1 2.1
~ g , ms 3.9
8.73 4.0%'
46.6. 3.6 4 P
138.9 5.2
108.7 2.7
8.52 3.84'
44.8 3.4 3.9b
144.2 4.6
106.7 3.0
.15
.16
.12
.19
.12
.49
1.6
2.7
2.1
EGTA Musion period CTR TRTA TRTB SEM
9.64 3.32b
3.7 5.2'
143.3 5 .O
108.6 2.9
35.5b
9.77 3.96' 40.8* 3.7 3.9b
143.1 5.5
106.8 3.0
9.36 4 . w
44.8' 3.7 4.3b
150.1 4.9
107.8 3.2
2 0 .ll
.07 24
.12
.83
.26
1.9
1.7
Period effect ** NS t NS NS NS NS NS NS
"bMeans with diffaent superscripts within period differ (P < .05). l c r ~ = control; TRT = mtment; EGTA = ettryiene gywi tetntacetate. %e DCAB for CTR, TRT A, aud TRT B were 338.35, and -127 mtq kgl of DM h eacalccmin period and 429,
tP < .lo. 68, and -147 meq kg-l of DM in EGTA infusion period.
**P < .01.
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CALCIUM KINETICS AND CATION-ANION BALANCE 423 1
TABLE 6. bbabi i ty of differences between mean rate constants (Ljj) and proportionality constants, (K1) for sheep fed diets with Merent cation-anion balanced diets @cAe) dariog a e u c a l d c and dariOg ethylene lywl tetraacetate W T A ) infasion period and the probability of diffaences in bj and K1 caused by the infusion. f
Probability of difference in meam between eacalcemic and EGTA
Probability of difference in means between different DCAB
constan? Eucalcemic EGTA Infusion infusion
Liz. d- 534 .862 .423 .826 .I37
.I22 L21, d-;
b. d-: .403 .607 .590
r, d;'
L32. .802 .a7 .590
L36 d- .644 .494 .004
L* d- Lf. d-' .649 .395 .%2
4 3 . d-, .393 ,321 .378
,567 .706 .0003 .oca .646 .OOO1
K1. h1 .833 .946 .710
to compartment i. lRate constants (Ljj) w h q j represents two different compartments and where Ca is transferred from compartment j
2h r, and = Rate constants for loss of radioisotope 45Ca to bone, to urine, and to the intestine f3om the system.
model because this would minimize the effect of the lag time without resorting to a c o r n - tion in the computer model for intestinal transit time. Because the physiological state of animals was changed experimentally @e., nor- mal or simulated lactation), it was necessary to use different rate constants &i> from one study to the next, even though two isotope studies were carried out in the same animals within a short period of time (2 wk). These rate constants were calculated based on our experimental data. In addition, the parameters representing loss of isotope from the system to the excreta (r, = intestine, and = urine) and to bone (Lo+) were changed in order to avoid inconsistencies in fitting the calculated model to actual data. proportionality constant is the equivalent of the calculated SA of plasma Ca at time zero; i.e., it is the reciprocal of the distribution in compartment 1 (Table 6). Data from sheep number 8 during the eucal-
cemic period were excluded before calculating a mean value because coprostasis developed during the last 3 d of the collection period.
Table 7 shows Ca inflow into and out of the exchangeable Ca pool (Va, V h VO-, VT, V,, V,, and Vd. Intake of Ca ranged from 7.6 to 16.7 g d-l and varied between blocks because the paired-feeding regimen was carried out
only within each block There was, however, no difference in mean intake of Ca among the dietary treatments. Regardless of dietary treat- ment, all sheep were in positive Ca balance in both periods, except for sheep number 2 that was fed the control diet during the EGTA infusion period (-37 g d-1). The total Ca
0 3 I I 8
Day
Figure 1. Cumulative isotope excretion in urine from eacalcemic or ethylene. glycol tetraacetate @GTA>infused sbbep fed diets having differeat cation-anion balances [+339. 6 8 , and -147 meq kg-' of DM in eucalcemic period and 429, +68, and -147 meq kg-' of DM in EGTA period for control and treatments (TR A and B, respectively] beginning with an injection of8, (d 0).
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4232 TAKAGI AND BLOCK
TABLE 7. Calcium intake and rates of Ca transport in sheep fed different cation-anion balanced @CAB) diets duriug eucalcemic and EGTA-infused
~bMeans within period with Merent SapasQiPts differ (P < .os). 'CTR = Control; TRT = treatment; J33TA = ethylene glycol teiraacetate. %%e DCAB for CLa, TRT A. and TRT B were 338, 35, and -127 mcq kg-l of DM in eacalcemic period and 429,
68, and -147 m q kg-' of DM in EGTA infaJim paiod. tP < .lo. 3va = Ca absorptiow V, = bone accretion; V, = bow rtsorptiow VT = V, + VO; Vf = fecal ~a excretio~; V, =
urinary Ca excre.tion.
inflow to the exchangeable pool or,> was composed entirely of intestinal absorption (VJ during both periods. Urinary excretion of Ca (Vu, percentage of
intake) was a f f d by dietary treatments dur- ing both periods in that both TRT A and TRT B (4.23 and 5.16% during the eucalcemic pe- riod and 17.2 and 15.6% during the EGTA infusion period, respectively) were higher than control (.18 and 13.2% during the eucalcemic and EGTA infusion periods, respectively). No other constants were diffemt during the eucal- cemic period.
During the EGTA infusion period, V, (grams per day), VO+ (percentage of in*), and V& (expressed as both grams per day and percentage of intake) were affected by the dietary treatments. Absorption of Ca (VJ by sheep fed TRT B (6.2 g d-l) was higher (P = .OS) than for TRT A (4.2 g el); however, no difference was observed when absorption was expressed as a percentage of intake (true Ca absorption rate). Bone accretion V, (loss of Ca to nonexchanging bone) expressed as a percentage of intake was higher (P = .04) in TRT A than TRT B (13.3 vs. 6.8%). but neither was different from control (8.31%).
The size of the total exchangeable Ca pool (MT) was not affected by DCAB nor by peri- ods, but Ca flux among the exchangeable Ca @ (R21-t Rl2 +R32 +R23 4- %3 +R34) af€e!cted by both treatments and periods. In the
TRT B, whereas both TRT A and TRT B caused higher Ca flux than the control during the EGTA infusion period.
Table 8 shows mean values of the eucal- cemic and EGTA infusion periods and @a- bility of differences. Simulating lactation by means of umtinuous EGTA infusion caused changes in Ca metabolism with a 48% reduc- tion in bone accretion V, (18.15 vs. 9.49%) and slight but signiscant increase in true ab- sorption rate (v, percentage of intake) (39.8 vs. 32.2%) compared with that of the eucal- cemic period. However, no difference was found in total Ca inflow to the exchangeable Ca pool (VT). mainly because of the lack of diffemce found in the other component of VT, V, The difference found in true absorption rate was reflected in a 24% reduction in endog- enous fecal excrehion (Vd. In gene& EcrrA infusion increased compartment mass and transfer rates between them except when
e u c a l d c period, ca flux was increased in
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CALCIUM KINETICS AND CATION-ANION BALANCE 4233
TABLE 8. Mcan compartment size and transfer rates for sheep during the encalcemic and EGTA' infusion periods and their pd!&ility of diffemnces.
'EGTA = Ethylene glycol tetraacetate. = ca absorption; V, = bone accretion; V, = bone resorption; VT = V, + v,; Vf = endogenous fwd Ca
excretion; Vu = uriuary Ca excretion.
animals were fed TRT B, in which case an increase in Ca flux associated with feeding reduced DCAB during eucalcemic period was observed.
DISCUSSION
It was shown from previous experiments (46,47) that manipulating DCAB, defined as milliequivalents [ma+ + K+) - (CY1 + S=)I, altered Ca metabolism. Negative DCAB that contained excesses of anions in relation to cations greatly increased urinary Ca excretion but did affect blood Ca concentration. This leads to the hypothesis that bone resorption must be increased in order to ensure normal extracellular Ca level. In this experiment, the kinetics of Ca metabolism in sheep fed M e r - ent DCAB were determined using the model of Ramberg et al. (40) and were fitted to a combi- nation of conventional Ca balance and radi- oisotope data using the computer programs CONSAM (5). Studies were carried out using normal (eucalcemic period) and EGTA-infused sheep @GTA infusion period). The latter was used as a model for simulated lactational Ca loss, thus allowing the comparison of kinetic changes in Ca caused by dietary treatments in a situation in which the Ca homeostatic mech- anisms are greatly and abruptly disturbed, i.e., at the onset of lactation. As previously reported (46), sheep fed
reduced and negative DCAB (TRT A and TRT
B, respectively) increased urinary excretion of Ca (V,,) compared with those fed controt how- ever, no changes were observed in Ca balance. Others (8, 48) also have observed hypercal- ciuria when feeding W C l to ruminants. They showed an increase in exchangeable Ca pool size (MT) that was associated with enhanced absorption from intestine, depressed bone ac- cretion (48), and enhanced bone resorption (8). Fredeen et al. (24) compared Ca kinetics of does (goats) fed an acidogenic diet supple- mented with HCl with does fed an alkalogenic diet supplemented with NaOH. Hypercalciuria and a reduction of bone accretion rate were found in goats fed acidogenic diet. Oral admin- istration of HCl to uraemic rats also resulted in an increase in the diversity of osteoclasts in bone and a reduction in bone mineralization rate (15).
Hypercalciuria observed in these studies and others may suggest that the effect is asso- ciated with a mild metabolic acidosis created by feeding acidogenic diets. However, some studies could not confirm the effect on bone metabolism (4). Thus, it was suggested that the effects of metabolic acidosis on bone metabo- lism become appatent after a long adaptation period, with rapidly growing young animals (52), or during periods of increased demand for Ca, such as late gestation and the onset of lactation (7, 35). Dietary acid stress provided by SO,, IWLQ, or both in rats showed ac-
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4234 TAKAGI AND BLOCK
celerated bone resorption only after 2 mo of adaptation (51) or in young animals with rapid bone turnover (33). Block (7) and Leclerc and Block (35) fed reduced rations, negative cation-anion balanced rations, or both to dry cows for 60 d and demonstrated that bone mobilization, reflected by the concentration of plasma-free hydroxyproline, increased during the p e r i p d e n t period. These mineral manipulations in the diet may, therefore, simu- late the direct addition of acids to the diet.
Previous kinetic studies demonstrated that an increased retention of Ca, resulting from
crease in bone resorption (V,) but little or no changes in bone accretion (Vw) in rats (13, 17) and in ruminants (11). Fuxthermore, cows that suffered from parturient hypocalcemia at the onset of lactation showed a temporal re- duction in outflow of Ca from bones (40). This suggested that bone resorption is an important feedback mechanism for Ca homeostasis (9). In our experiment, no correlation was observed between intake or retention of Ca and bone accretion rate (r = .03 and .lo, respectively), but a strong negative correlation was found between bone mobilization rate and intake of Ca (r = -.37, P = .07) and between bone mobilization rate and Ca balance (r = -.53, P = .008).
In some studies, the total M o w to compart- ment 1 (VT) was exceeded by the calculated value for Ca absorbed from the intestine (VJ. Consequently, some negative values for Ca mobilization from bone (V,) were obtained by calculating V, = VT - V,. Negative values of Vo- also were reported in cows and rats fed relatively high dietary Ca (1.4% DM) (13, 41) and in cows prior to the onset of lactation (40). The inherent errors of balance techniques would increase as dietary Ca intake increases, and most of the error involved small cumula- tive losses in feces and urine and incomplete measurements of intake, both of which favour a positive balance and might lead to overesti- mation of VT, underestimation of V,, or both. However, these possible emrs could not ac- count for the magnitude of the negative V, values that were observed in our trial (i.e., TRT B during the E T A infusion period). Some fluctuation in daily fecal output was observed with minimum changes in feed intake during the 7-d collection period. Even though
feeding increasing dietary Ca, led to an in-
Ca retention within the gastrointestinal tract is horn to be minimal (2), a long digesta transit time in ruminants compared with that of non- ruminants, with uneven excretion rates, might lead to overestimation of Ca balance. Some implications of negative values of V s have been discussed by Bronner (12). One of the possible explanations is that a fiaction of the absorbed Ca may be deposited into bone with- out equilibrating with al l of the Ca in compart- ment 1, implying the existence of nonexchang- ing or slowly exchanging fraction of the blood Ca that cannot be traced by intravenous injec- tion of isotope (45Ca) (50). Slowly exchanging fractions of blood Ca have been reported in some species, but not in ruminants (40). How- ever, even in ruminants, some indirect evi- dence based on the dissimilarity of the SA of urine or milk and plasma Ca (26,50) suggests that existence of such a fraction is plausible. Unphysiological negative values of bone mobilization rates (V,) observed in some of the animals in our study suggest that this model might not be applicable for Ca kinetic studies, in which a large difference in the level of dietary Ca exists. In our study, however, variation of the level of Ca in experimental diets was relatively small (.88 to 1.05% DM). Therefore, the utilization of a four compart- ment model to investigate the effect of manipulating DCAB on Ca kinetics is justified in this case.
In our experiment, the total exchangeable pool sizes of Ca (MT) in the eucalcemic period were approximately three to four times larger than those in sheep mea& by kinetic studies using a two compartment model (10, 11,30). The MT were 70% larger than those of mature, nonlactating cows determined by the four compartment model as used in this experi- ment (40). Values were higher because of rela- tively higher VT and Vw, which is a reflection of high dietary Ca intake in this experiment compared with that of Ramberg et al. (41) (.30 vs. .175 g of Ca kg' of BW). This relatively high dietary intake of Ca, ranging from 11 to 13 g d?, compared with that of requirements (7.7 g bl) probably is attributed to small changes in MT between the eucalcemic and the EGTA infusion periods.
There was no difference in the size of total exchangeable Ca pool among dietary treat- ments during either period, This is contrary to
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CALCIUM KINETICS AND CATION-ANION BALANCE 4235
results of Vagg and Payne (48) but confirmed the results of Fredeen et al. (25), who also hypothesized that size of the exchangeable Ca pool is influenced only by the action of parathyroid hormone (PTH), the secretion of which is triggered by a reduction of blood ionized form of Ca (Ca*) (21). A positive correlation between plasma PTH and the size of exchangeable Ca pool was observed in non- pregnant nonlactating cows (41) and in EDTA- infused hypocalcemic cows (39). Increased plasma Ca* concentrations were observed in animals fed reduced DCAB (TRT A and TRT B) in our experiment. Such should result in a corresponding suppression of F”H secretion, leading to a reduction in the size of exchangea- ble Ca pool. The discrepancy between the two findings may be caused by alteration in the acid-base status of animals. Bushinsky et al. (14) created metabolic acidosis in rats by feed- ing IW&l and found an increased serum Ca* concentration but could not detect a reduction in plasma PTH concentration. Therefore, direct effects or consequences of disturbing acid-base balance of an imals toward acidosis by feeding reduced DCAB may alter the relationship be- tween F’”H secretion and plasma Ca* concen- trations.
It is well established that endogenous fecal Ca (Vf) is affected by DMI and not by dietary intake of Ca (9). Small variations observed in this experiment also agree with previous find- ings (40). The Vf represents the amount of Ca that was secreted into the intestine and not reabsorbed. A 24% reduction in Vf observed in EGTA-infused lambs may reflect either a decrease in secretion of Ca into the intestine or a more efficient reabsorption of the en- dogenously secreted gastrointestinal Ca. The latter is more consistent with the change ob- served in true Ca absorption rate (V, percent- age of intake). A positive correlation (r = .64, P = .007) was observed between Vf and Fp (total fecal Ca excretion), indicating that en- dogenously secreted and dietary Ca were mixed within the intestine and that no dis- crimination in the reabsorption process oc- m e d .
Simulated loss of Ca in lactation created by continuous E T A infusion resulted in transient responses in the Ca homeostatic mechanism, which reflected the kinetics of the feedback signals involved in the control of plasma and
bone Ca Included in these pathways are the kinetic parameters associated with secretion, metabolism, and action of feedback modulators such as FTH, calcitonin, and 1,25- dihydroxyvitamin D3 and the effect of those on their cellular targets in intestines, kidneys, and bones. During this period, effects of reducing DCAB on Ca kinetics were significant. Elevated intestinal absorption of Ca (VJ with a concomitant decrease in bone accretion (Vm) to accommodate an increase in Ca demand was observed in cows (42) and ewes (9). However, there is no hormonal study available in the literature to illustrate effects of manipulating DCAB.
Several studies indicated that prepartum in- take of acidogenic and alkalogenic components of the diet is more important than the level of dietary Ca with regard to the incidence of hypocalcemic parturient paresis (milk fever) (7, 18, 20). Conventionally, a high intake of dietary Ca is considered to be one of the most important predisposing factors to milk fever (28). However, when an acidogenic diet was fed to prepartum cows predisposed to milk fever, it was observed that relatively high die- tary Ca may be beneficial (1 8, 20,49). Ander- son et al. (1) found that feeding elevated die- tary Ca enhanced absorption, retention, and depressed bone resorption but had no effects on the exchangeable Ca pool size, which forcsd animals to become severely hypocal- cemic at parturition (41). Therefore, it was suggested that the size of the exchangeable Ca pool in preparturient cows may be of primary importance. Fredeen et al. (25) demonstrated a beneficial effect of feeding acidogenic diets on Ca homeostasis to recover from sudden loss of Ca via manipulation of acid-base status of the animal. In contrast with our study, mild meta- bolic acidosis imposed by acidogenic diets elevated exchangeable Ca pool (48). Further- more, incidence of milk fever is dramatically increased when diets are ahlogenic and reduced when diets are acidogenic (7, 18, 20). In our study, hypercalciuria caused by feeding reduced DCAB, negative DCAB, or both caused sheep to eliminate excess Ca that was absorbed, resulting in no change in Ca reten- tion; animals increased or maintained high flux through the exchangeable Ca pool. This was achieved without the inhibitory effects on bone resorption observed when alkalogenic diets
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4236 TAKAGI AND BLOCK
with relatively high dietary Ca levels were fed
Our experiment indicated that feeding reduced DCAB to sheep resulted in an in- creased Ca flux through the exchangeable Ca pool but had no effects on the size of the Ca pool. This increased flux was maximized when Ca demand of animals was increased by s h u - lated lactational Ca loss caused by continuous infusion of S T A . Even though concentration of total plasma Ca was not significantly af- fected by DCAB , plasma ionized Ca++ concen- trations were elevated by feeding reduced DCAB. This indicated that there was a change in the interrelationship between the Ca- regulating hormones (FTH, vitamin D, and cal- citonin), responsiveness to target organs (bone, kidney, and intestine), or both. Hypercalciuria associated with reduced DCAB with corres- ponding increases in intestinal Ca absorption and in bone resorption may play an important role in sustaining a high level of Ca flux through the exchangeable Ca pool, especially in situations of high Ca demand such as post- parturient hypocalcemia.
(24).
ACKNOWLEDGMENTS
The authors acknowledge and appreciate the assistance of Allan Fredeen of the Nova Scotia Agricultural College for his contributions in the analysis of the models used.
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