NUTRITION, FEEDING, AND CALVES Potentiometric Determination of Ionized and Total Calcium in Blood Plasma of Holstein and Jersey Cows H. T. BALLANTINE and J. H. HERBEIN' Department of Dairy Science Virginia Polytechnic Institute and State University Blacksburg 24061 ABSTRACT
The effects of stage of lactation and breed on total and ionized calcium in
blood plasma were determined for eight Holstein and eight Jersey cows fed a diet containing .25% Ca during the dry period and fed a diet with SO% Ca after calving. Blood samples were obtained by jugular venipuncm at 2-wk intervals from 4 wk before Calving through wk 10 Of lactation. Total and ionized Ca contents of fresh plasma were determined potentiometrically with an ion-specific electrode. Total Ca in plasma also was deterby atomic absorption mined spectrophotometry. Average concentrations determined by atomic absorption were approximately .7 mgldl higher than those for the ion-specific electrode. Plasma total and ionized Ca were lower in both breeds at calving than any other time during the sampling period. Jersey cows had lower total Ca (7.47 mg/dl) and ionized Ca (4.25 mg/dl) than Holsteins (8.10 and 4.66 mg/dl) on day of calving. Ionized Ca as a percentage of total calcium increased from 55 to 57% at parturition and then decreased slowly to 53% at peak lactation. Changes in percentage of ionized Ca may indicate the contribution of bone resorption relative to dietary Ca in meeting the Ca requirement for lactation. (Key words: plasma calcium, ionized calcium, parturition)
Abbreviation key: AAS = atomic absorption spectrophotometxy, ICA = ionized calcium, NORMCA = normalized calcium, TCA = total calcium. IKTRODUCTION
Calcium is a critical component of many physiological processes including bone metabolism, blood clotting, nerve impulse transmission, muscle contraction, and milk synthesis. Homeostatic mechanisms maintain blood Ca concentration within a range of 9 to 11 mg/dl (7, 9). Blood Ca concentration usually is reported as total Ca, but it can be partitioned into three distinct fractions (10). Approximately 45% of the total is bound to plasma proteins, primarily albumin. A small amount (5%) is in the ionic form associated with low molecular weight compounds such as lactate, citrate, or bicarbonate. The remainder is free Ca ions, which are readily available for metabolic functions. Total Ca concentration may give an overall indication of Ca status, but ionized Ca concentration may be a better estimate of the amount available for uptake and utilization by tissues. Inability of dairy cows to maintain Ca homeostasis at parturition was indicated by varying degrees of hypocalcemia that depended on prepartum dietary Ca intake, breed, age, and parathyroid hormone injection (1, 3, 4, 7, 9). The studies were designed to enhance susceptibility to parturient hypocalcemia and to evaluate endocrine responses or various means of preventing hypocalcemia (parturient paresis). However, total Ca Concentration in plasma or serum were not determined until the end of the trials using stored samples. A rapid method for determination of blood Ca concentration folReceived June 1. 1990. lowing pamuition would be beneficial in such Accepted Augast 17,1990. trials to monitor Ca status continuously. 'Correspondence: 3270 Litton-Raves Building, Potentiometric instruments with Ca-specific Department of Dairy Scimce, V i a Polytechnic Institute electrodes offer a means for rapid analysis of aad State University, Blacksburg 24061. 1991 J Dairy Sci 74:446-449
446
PLASMA IONIZED A M ) TOTAL CALCNM
total and ionized Ca in whole blood, plasma, or serum of humans (10). A recent clinical study (5) indicated the validity and usefulness of potentiometric methods to determine ionized Ca in blood of dairy cows. Our objective was to evaluate the effects of stage of lactation and breed (Holstein versus Jersey) on plasma concentrations of total and ionized Ca determined by a potentiometric Ca analyzer. Selected plasma samples also were used to determine total Ca content by atomic absorption spectrophotometry.
447
this study was between 7.5 and 7.6 at time of analysis. Aliquots of plasma from samples taken from 4 wk before to 4 wk after parturition were frozen at -20'C for later analysis of TCA by atomic absorption spectrophotometq
(9).
The diet fed during the prepartum period consisted of 80% chopped orchardgrass hay and 20% corn silage (DM basis). Each cow received 1.14 kg/45 kg of BW. The diet contained .25% Ca. After calving, all cows were fed a diet containing 28.8% corn silage, 22.4% alfalfa haylage, and 48.8% concentrate. Chemical composition of the diet was 16.4% CP, MATERIALS AND METHODS 23.3% ADF, and 1.82% ether extract. The diet Eight Holstein and eight Jersey cows were contained SO% Ca, .39% P, and .24% Mg. After we determined that no significant selected randomly to obtain blood samples during the lactation cycle. AU cows were entering (Pe.05) effects had occurred due to time of their third or later lactation. Cows were housed sampling within a day, averages for the samples in a tie-stall barn throughout the study. taken at 0800,1600, and oo00 h were used for Heparinized blood samples were obtained by further statistical analysis. To measure week of jugular venipuncture at 2-wk intervals from sampling and breed effects, data were analyzed approximately 4 wk before calving to wk 10 of as a split-plot design using the general linear lactation. Sampling times within day were models procedure of SAS (8). Breed was tested 0800,1600, and oo00 h. Heparinized blood was for significance using cow within breed mean kept on ice until plasma was separated by square error. Week and breed by week interactions were tested for significance using residual centrifugation at 3200 x g for 15 min. Plasma was aspirated immediately into a error. Differences were considered significant Nova 7 calcium analyzer (Nova Biomedical, at Pe.05. Waltham, MA), which determines total Ca (TCA), ionized Ca (ICA), and normalized Ca RESULTS AND DISCUSSION (NORMCA) and pH. Nomalized Ca ion conAs expected, plasma TCA determined by the cenlration is ICA corrected to pH 7.4 (10). The Ca-specific electrode was lower at calving (wk instrument calculates NORMCA using the 0) than any of the other weeks (Table 1). The equation: degree of hypocalcemia at calving was greater in Jerseys than in Holsteins. The ICA and log C 3 7 = l o g c a r - .24 (7.4 - x) NORh4CA followed the same trend as plasma TCA. No significant differences in plasma pH where: (at time of analysis) due to breed or week of sampling were found. Both ICA and NORMCA x = pH of sample; are listed to emphasize the extent of correction c< = ICA concentration in test solution; for sample pH above 7.4. and At calving, two Jerseys had TCA below 5.0 t t = normalized concentration of ICA at mg/dl and had to be treated for paaurient parec3'4 pH = 7.4. sis. None of the Holsteins displayed clinical symptoms of hypocalcemia. Littledike (6) re(Key words: Exposure of blood to air during ported that the incidence of parturient paresis in processing causes pH to increase slightly due to the Jersey breed was 12.4 to 30% compared loss of C% and a shift in C@-bicarbonate with 2.1 to 3.9% in the Holstein breed. The equilibrium The equilibrium shift favors the incidence rate was 25% for Jerseys in this study protein-bound form Of Ca resulting in lower despite their low Ca diet prior to calving. PreICA readings. The pH of all plasma samples in partum low Ca diets prevented parturient pareJoamal of Dairy Science Vol. 74, No. 2, 1991
BALLANTINEANDHERBEIN I
I
I
I
I
0Atomic
Absorption Ion Electrode
I
I
I
I
I
-4
-2
0
2
4
WEEKS
Figure 1. Total calcium in plasma of multiparous Holstein and Jersey alws by weeks prepartum and postpamm, as determinad by atomic absorption spectrophotometry and potentiometric (ion electrode) calcium analyzer. Vertical error bars indicate SEM (n = 16).
sis in some studies (2, 3, 10). but plasma TCA below 8 mg/dl was noted at parhlrition in most cows. In general, TCA determined by the potentiometric Ca analyzer appeared to be similar to TCA concentrations determined by atomic absorption spectrophotometry ( U S ) at corresponding stages of lactation in other studies (1, 3, 9, 11). However, comparison of methods
used in this study indicated that concentrations of plasma TCA determined potentiometrically were lower than those determined by AAS Figure 1). Watson and Anbar (10) reported 3% lower means when these r;iethods were compared using human plasma. Our means averaged 7% lower with less discrepancy below 9 mg/dl than above. The discrepancy at higher Ca concentrations is not unique to bovine plasma: we obtained similar results for ovine, equine, porcine, chicken, and turkey plasma (data not presented). We tried various methods to account for the incomplete recovery above 9 mg/ dl, but no practical solution was apparent. Determination of ICA is pHdependent. Alteration of the sample in any manner seemed to interfere with reliable determination of pH and, thus, ICA. Sample dilution, for example, provided a better correlation with AAS estimates of TCA, but ICA data are invalid. The dilution procedure is recommended by the manufacturer for urine and rumen fluid with the warning that the only valid readout is TCA. The NORMCA as a percentage of TCA was calculated for each week Breeds did not differ significantly at any week. Data for breeds were combined and plotted in Figure 2. The percentage of NORMCA was higher at calving than at wk 2 and 4 prepartum. The higher percentage at parturition coincides with pronounced increases in plasma parathyroid hormone (4, 9) and 1.25-dihydroxyvitamin D (1, 3) concentrations. Ehhanced mobilization of Ca from bone due to these endocrine stimuli at calving may have
TABLE 1. Least square.s means for plasma total calcium (TCA), ionized calcium QCA), and normalized calcium (NORMCA) by waeb prepartum and postpartum for multiparous Holstein @ andI Jersey ) (I) cows.
TCA'
NORMCA~
ICA'
Week
H
J
H
4 -2 0 2 4 6 8 10
9.40 9.71 8.10" 9.12 9.16 9.60 9.33 9.35 .18
9.05 9.55 7.47 8.94 9.03 9.34 8.97 922 .18
4.67 4.80 4.16, 4.63 4.58 4.68 453 4.56
J
H
J
4.54 4.70 3.78 453 457 4.62 4.38 4.49
5.20 5.37 4.66. 523 5.10 5.18 5 .oo 5.07
4.92 5.27 4.25 5.09 5.06 5.08 4.84 4.86
.08
.10
.10
(ddo
SEM
.as
'Concentrations determined by a Nova 7 calcium analyzer. +Significant breed difference (P
The effects of stage of lactation and breed on total and ionized calcium in
blood plasma were determined for eight Holstein and eight Jersey cows fed a diet containing .25% Ca during the dry period and fed a diet with SO% Ca after calving. Blood samples were obtained by jugular venipuncm at 2-wk intervals from 4 wk before Calving through wk 10 Of lactation. Total and ionized Ca contents of fresh plasma were determined potentiometrically with an ion-specific electrode. Total Ca in plasma also was deterby atomic absorption mined spectrophotometry. Average concentrations determined by atomic absorption were approximately .7 mgldl higher than those for the ion-specific electrode. Plasma total and ionized Ca were lower in both breeds at calving than any other time during the sampling period. Jersey cows had lower total Ca (7.47 mg/dl) and ionized Ca (4.25 mg/dl) than Holsteins (8.10 and 4.66 mg/dl) on day of calving. Ionized Ca as a percentage of total calcium increased from 55 to 57% at parturition and then decreased slowly to 53% at peak lactation. Changes in percentage of ionized Ca may indicate the contribution of bone resorption relative to dietary Ca in meeting the Ca requirement for lactation. (Key words: plasma calcium, ionized calcium, parturition)
Abbreviation key: AAS = atomic absorption spectrophotometxy, ICA = ionized calcium, NORMCA = normalized calcium, TCA = total calcium. IKTRODUCTION
Calcium is a critical component of many physiological processes including bone metabolism, blood clotting, nerve impulse transmission, muscle contraction, and milk synthesis. Homeostatic mechanisms maintain blood Ca concentration within a range of 9 to 11 mg/dl (7, 9). Blood Ca concentration usually is reported as total Ca, but it can be partitioned into three distinct fractions (10). Approximately 45% of the total is bound to plasma proteins, primarily albumin. A small amount (5%) is in the ionic form associated with low molecular weight compounds such as lactate, citrate, or bicarbonate. The remainder is free Ca ions, which are readily available for metabolic functions. Total Ca concentration may give an overall indication of Ca status, but ionized Ca concentration may be a better estimate of the amount available for uptake and utilization by tissues. Inability of dairy cows to maintain Ca homeostasis at parturition was indicated by varying degrees of hypocalcemia that depended on prepartum dietary Ca intake, breed, age, and parathyroid hormone injection (1, 3, 4, 7, 9). The studies were designed to enhance susceptibility to parturient hypocalcemia and to evaluate endocrine responses or various means of preventing hypocalcemia (parturient paresis). However, total Ca Concentration in plasma or serum were not determined until the end of the trials using stored samples. A rapid method for determination of blood Ca concentration folReceived June 1. 1990. lowing pamuition would be beneficial in such Accepted Augast 17,1990. trials to monitor Ca status continuously. 'Correspondence: 3270 Litton-Raves Building, Potentiometric instruments with Ca-specific Department of Dairy Scimce, V i a Polytechnic Institute electrodes offer a means for rapid analysis of aad State University, Blacksburg 24061. 1991 J Dairy Sci 74:446-449
446
PLASMA IONIZED A M ) TOTAL CALCNM
total and ionized Ca in whole blood, plasma, or serum of humans (10). A recent clinical study (5) indicated the validity and usefulness of potentiometric methods to determine ionized Ca in blood of dairy cows. Our objective was to evaluate the effects of stage of lactation and breed (Holstein versus Jersey) on plasma concentrations of total and ionized Ca determined by a potentiometric Ca analyzer. Selected plasma samples also were used to determine total Ca content by atomic absorption spectrophotometry.
447
this study was between 7.5 and 7.6 at time of analysis. Aliquots of plasma from samples taken from 4 wk before to 4 wk after parturition were frozen at -20'C for later analysis of TCA by atomic absorption spectrophotometq
(9).
The diet fed during the prepartum period consisted of 80% chopped orchardgrass hay and 20% corn silage (DM basis). Each cow received 1.14 kg/45 kg of BW. The diet contained .25% Ca. After calving, all cows were fed a diet containing 28.8% corn silage, 22.4% alfalfa haylage, and 48.8% concentrate. Chemical composition of the diet was 16.4% CP, MATERIALS AND METHODS 23.3% ADF, and 1.82% ether extract. The diet Eight Holstein and eight Jersey cows were contained SO% Ca, .39% P, and .24% Mg. After we determined that no significant selected randomly to obtain blood samples during the lactation cycle. AU cows were entering (Pe.05) effects had occurred due to time of their third or later lactation. Cows were housed sampling within a day, averages for the samples in a tie-stall barn throughout the study. taken at 0800,1600, and oo00 h were used for Heparinized blood samples were obtained by further statistical analysis. To measure week of jugular venipuncture at 2-wk intervals from sampling and breed effects, data were analyzed approximately 4 wk before calving to wk 10 of as a split-plot design using the general linear lactation. Sampling times within day were models procedure of SAS (8). Breed was tested 0800,1600, and oo00 h. Heparinized blood was for significance using cow within breed mean kept on ice until plasma was separated by square error. Week and breed by week interactions were tested for significance using residual centrifugation at 3200 x g for 15 min. Plasma was aspirated immediately into a error. Differences were considered significant Nova 7 calcium analyzer (Nova Biomedical, at Pe.05. Waltham, MA), which determines total Ca (TCA), ionized Ca (ICA), and normalized Ca RESULTS AND DISCUSSION (NORMCA) and pH. Nomalized Ca ion conAs expected, plasma TCA determined by the cenlration is ICA corrected to pH 7.4 (10). The Ca-specific electrode was lower at calving (wk instrument calculates NORMCA using the 0) than any of the other weeks (Table 1). The equation: degree of hypocalcemia at calving was greater in Jerseys than in Holsteins. The ICA and log C 3 7 = l o g c a r - .24 (7.4 - x) NORh4CA followed the same trend as plasma TCA. No significant differences in plasma pH where: (at time of analysis) due to breed or week of sampling were found. Both ICA and NORMCA x = pH of sample; are listed to emphasize the extent of correction c< = ICA concentration in test solution; for sample pH above 7.4. and At calving, two Jerseys had TCA below 5.0 t t = normalized concentration of ICA at mg/dl and had to be treated for paaurient parec3'4 pH = 7.4. sis. None of the Holsteins displayed clinical symptoms of hypocalcemia. Littledike (6) re(Key words: Exposure of blood to air during ported that the incidence of parturient paresis in processing causes pH to increase slightly due to the Jersey breed was 12.4 to 30% compared loss of C% and a shift in C@-bicarbonate with 2.1 to 3.9% in the Holstein breed. The equilibrium The equilibrium shift favors the incidence rate was 25% for Jerseys in this study protein-bound form Of Ca resulting in lower despite their low Ca diet prior to calving. PreICA readings. The pH of all plasma samples in partum low Ca diets prevented parturient pareJoamal of Dairy Science Vol. 74, No. 2, 1991
BALLANTINEANDHERBEIN I
I
I
I
I
0Atomic
Absorption Ion Electrode
I
I
I
I
I
-4
-2
0
2
4
WEEKS
Figure 1. Total calcium in plasma of multiparous Holstein and Jersey alws by weeks prepartum and postpamm, as determinad by atomic absorption spectrophotometry and potentiometric (ion electrode) calcium analyzer. Vertical error bars indicate SEM (n = 16).
sis in some studies (2, 3, 10). but plasma TCA below 8 mg/dl was noted at parhlrition in most cows. In general, TCA determined by the potentiometric Ca analyzer appeared to be similar to TCA concentrations determined by atomic absorption spectrophotometry ( U S ) at corresponding stages of lactation in other studies (1, 3, 9, 11). However, comparison of methods
used in this study indicated that concentrations of plasma TCA determined potentiometrically were lower than those determined by AAS Figure 1). Watson and Anbar (10) reported 3% lower means when these r;iethods were compared using human plasma. Our means averaged 7% lower with less discrepancy below 9 mg/dl than above. The discrepancy at higher Ca concentrations is not unique to bovine plasma: we obtained similar results for ovine, equine, porcine, chicken, and turkey plasma (data not presented). We tried various methods to account for the incomplete recovery above 9 mg/ dl, but no practical solution was apparent. Determination of ICA is pHdependent. Alteration of the sample in any manner seemed to interfere with reliable determination of pH and, thus, ICA. Sample dilution, for example, provided a better correlation with AAS estimates of TCA, but ICA data are invalid. The dilution procedure is recommended by the manufacturer for urine and rumen fluid with the warning that the only valid readout is TCA. The NORMCA as a percentage of TCA was calculated for each week Breeds did not differ significantly at any week. Data for breeds were combined and plotted in Figure 2. The percentage of NORMCA was higher at calving than at wk 2 and 4 prepartum. The higher percentage at parturition coincides with pronounced increases in plasma parathyroid hormone (4, 9) and 1.25-dihydroxyvitamin D (1, 3) concentrations. Ehhanced mobilization of Ca from bone due to these endocrine stimuli at calving may have
TABLE 1. Least square.s means for plasma total calcium (TCA), ionized calcium QCA), and normalized calcium (NORMCA) by waeb prepartum and postpartum for multiparous Holstein @ andI Jersey ) (I) cows.
TCA'
NORMCA~
ICA'
Week
H
J
H
4 -2 0 2 4 6 8 10
9.40 9.71 8.10" 9.12 9.16 9.60 9.33 9.35 .18
9.05 9.55 7.47 8.94 9.03 9.34 8.97 922 .18
4.67 4.80 4.16, 4.63 4.58 4.68 453 4.56
J
H
J
4.54 4.70 3.78 453 457 4.62 4.38 4.49
5.20 5.37 4.66. 523 5.10 5.18 5 .oo 5.07
4.92 5.27 4.25 5.09 5.06 5.08 4.84 4.86
.08
.10
.10
(ddo
SEM
.as
'Concentrations determined by a Nova 7 calcium analyzer. +Significant breed difference (P
