Endogenous nature of circadian in calcium metabolism JEAN-FRANCOIS AND GERARD
STAUB, MILHAUD
Service de Biophysique,
ANNE-MARIE
CHU Saint-Antoine,
ANNE-MARIE PERAULT-STAUB, STAUB, JEAN-FRAN~OIS, AND GERARD MILHAUD. Endogenous nature of circadian rhythms in calcium metabolism. Am. J. Physiol. 237(5): R3llR31’7, 1979 or Am. J. Physiol.: Regulatory Integrative Comp. Physiol. 6(3): R311-R317, 1979.-Rats studied when the lights are on from 0600 to 1800 daily and fed only in the dark period displayed circadian rhythms in plasma calcium (ionized and total) and 45Ca concentrations, 6 and 8 days after 45Ca administration. In rats fed a calcium-deficient diet, the amplitude of daily variation of plasma ionized and total calcium increased remained esmarkedly whereas plasma 45Ca daily fluctuation sentially unchanged. In the calcium-deficient rats, significant correlations between plasma calcium and 45Ca and between plasma calcium and magnesium were observed throughout the 24 h; circadian periodicity of calcium metabolism persisted in rats fasted overnight, regardless of the illumination schedule. Normal daily fluctuations in plasma 45Ca, lost after thyroparathyroidectomy (TPTX), were restored by feeding the TPTX rats a high-calcium diet. These results demonstrate clearly that circadian rhythms of calcium metabolism occurred irrespective of the light-dark schedule, the calcium supply through intestines and the thyroparathyroid system. An attractive suggestion is that circadian rhythmicity originates as a result of dynamic properties involving nonlinear processes of calcium metabolism. rat; calcium
homeostasis;
dissipative
structure
INVESTIGATORS have demonstrated the existence of circadian rhythms that regulate calcium metabolism in the rat. These studies include observations on the circadian rhythms in total (7, 19) and ionized plasma calcium (4)) urinary calcium excretion (2)) serum calcitonin (M), and metabolic activity of bone (16). In some experiments in rats, the patterns of the daily fluctuations both in plasma calcium and its radionuclide concentration resulting from the administration of 45Ca 1 wk before the experiments were measured (11, 19). All of these data, favoring the existence of “internal periodic changes” in calcium homeostatic processes that could be correlated with the periodic environmental events, led us to postulate a new concept for calcium homeostasis (12), namely that rhythmicity must be a fundamental requisite for normal calcium metabolism. The aim of the present investigation was to obtain further insight into the origin and the role of the internal circadian processes in calcium regulation. The daily fluctuations in plasma calcium and 45Ca concentrations were used as measures of the calcium metabolism processes in rats fed an essentially calcium-free diet where the usual
MANY
0363~6119/79/~-~$01.25
Copyright
0 1979 the American Physiological
rhythms
PERAULT-STAUB, 75571 Paris Ce’dex 12, France
periodic calcium supply through the intestinal absorption is absent. This study also included the effect on circadian rhythms of a 24-h fast either in the dark or in the usual light-dark schedule, and of thyroparathyroidectomy with or without access to a high-calcium diet. The results in this report emphasize the need to consider rhythmicity as a primordial endogenous characteristic of the physiological control of calcium metabolism; the results also help clarify the role of these endogenous regulatory processes in calcium homeostasis. METHODS
Animals Weanhng male Wistar C.F. rats were placed in special cages adapted for automatic control of lighting and access to food. The rats were exposed to darkness (1800-0600) on a 12-h light-dark (LD) schedule. Food was available only during the dark period; water was provided ad libitum throughout the 24 h.
Diets The semisynthetic diet (U.A.R., Villemoisson/orge, France) contained 0.7% phosphorus; calcium carbonate was added to reach the desired level. The calcium-deficient regimen was essentially free of calcium (less than 0.03%). The normal diet contained 0.7% calcium and the high-calcium diet, 1.5% calcium. The diets contained adequate amounts of vitamin D3 and necessary minerals.
Protocols Effect of calcium-deficiency. EXPERIMENT A. During the 1st postweanhng wk, 80 animals were fed a normal diet and drank tap water; they were then divided into two equal groups. During the next 3 wk, the experimental group was restricted to a calcium-deficient diet and distilled water, and the control group was fed a normal diet and tap water. Two weeks later the rats were weighed, individually stamped, randomly allocated into subgroups of five rats each, and injected intravenously with radioactive calcium (100 PCi of 45Ca as CaC12). At selected times during the day (0600, 1000, 1400, 1800, 2200, or 0200) rats in the subgroups were weighed and blood samples (0.1 ml) were obtained by retro-orbital puncture. The blood was collected in heparinized tubes and centrifuged immediately. On the 8th day, a larger blood sample (1.5 ml) was obtained by cardiac puncture in a lightly Society
It311
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (139.184.014.150) on August 11, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
R312
STAUB,
heparinized syringe; the syringe was centrifuged immediately, the piston down. An aliquot of the plasma sample was used immediately to measure ionized calcium, the remainder was used to determine total calcium. EXPERIMENT B. The effect of calcium deficiency on plasma magnesium was studied in a separate experiment. The experimental group had access to a calcium-deficient diet and distilled water for 18 days; the rats in the control and experimental groups were bled by retro-orbital puncture only once during one 24-h period. Effect of fasting. Weanling rats were fed a normal diet and received tap water ad libitum for 3 wk; they had access to food only during the dark period on a 12-h LD schedule. Radioactive calcium (100 &i of 45Ca as CaC12) was administered 1 wk before the first blood sample was obtained. At 1800 on the experimental day, one group was allowed the usual access to food and the 12-h LD schedule; for the two other groups, food and the sawdust litter were removed, distilled water was available and the illumination schedule was either unchanged (second group) or replaced by constant darkness (third group). Rats, 5 to 10 per subgroup, were bled only once by retroorbital puncture at 4-h intervals during any one 24-h period. Effect of thyroparathyroidectomy. Parathyroidectomy was performed by blunt dissection under ether anesthesia on 2-mo-old rats. (Animals were considered successfully parathyroidectomized if the plasma calcium was less than 8.0 mg/lOO ml 2 days after the operation.) Five days later, the animals previously maintained on the regular diet were distributed into two groups of 40 rats each. One group was maintained on the normal diet, the other was placed on a high-calcium diet. The radioactive calcium was administered (100 @i of 45Ca as CaC12). Five days later, the rats were thyroidectomized. Experimental blood samples were collected 2 days after thyroidectomy, at a time when the deprivation of endogenous iodinated hormones was not apparent. Thyroparathyroidectomized rats (TPTX) six to seven per subgroup, were bled by retro-orbital puncture at 4-h intervals; the rats were only bled one time during a 24-h period.
PERAULT-STAUB,
AND
MILHAUD
RESULTS
Total Plasma Calcium and Magnesium Control and Calcium-Deficient Rats
in
Figures 1 and 2 show the levels of the total plasma calcium in control and calcium-deficient rats, (expt A). Three factors were considered: 1) the mean 24-h value, 2) the amplitude of the daily variation, and 3) the pattern of the daily fluctuation. On the 1st day of the experiment (after 2 wk on the calcium-deficient diet) the mean 24-h plasma calcium had fallen to the level of 9.01 t 0.14 mg/lOO ml (Fig. 2) compared to 10.99 t 0.07 mg/lOO ml in control rats (Fig. 1). The difference was highly significant (P < 0.001). The mean value in calcium-deficient rats continued to de-
mean zlh-vsluo day O-l - l-2 - 3-4 - 5-6 (SO) ” 7-0
(341 (341
(60)
(50)
(mg/lOOml) 10.99 +0.07 11.22 to.07 11.07 zo.04 11.10 r0.09 11.14 TO.06
(32)
t (3s)
FIG. 1. Circadian variation in total plasma calcium of control rats. Pattern of 24-h variation was highly reproducible from 1 day to the next. Data, means & SE, were pooled over the &day period. Numbers in parentheses indicate number of samples used for estimation of mean 5-day value at each selected time. Heavy horizontaz bar represents time of feeding and darkness. Preprandial fall between 1400 and 1800 was significant, P = 0.002. Mean 24-h values of total plasma calcium on each of 5 days of experimental week are listed on right.
Blood Analysis
Plasma total calcium and magnesium were determined by atomic absorption spectrophotometry (Perkin-Elmer model 503). Plasma ionized calcium concentration was measured anaerobically with model SS-20 ionized calcium analyzer (Orion Research, Cambridge, MA). Plasma radioactivity was measured in a liquid scintillation counter, corrected for the daily body weight to eliminate individual fluctuations and expressed as dpm/ml. moan 24hualu:
Statistical
Treatment
Rats were assigned randomly into experimental and treatment groups. The data from each experiment were subjected to analysis of variance, and standard error of the means (SE) were calculated. Statistical analysis between groups was performed by Student’s t test; P < 0.05 was considered as significant. Correlation coefficients (r ) between parameters were calculated from the individual values bv the method of linear least sauares.
0 9.Ol to.14 8.63tO.14
8.43zO.13
7.98?0.14
7.42:0.19(mg /lOOml )
2. Circadian variation of total plasma calcium in calcium-deficient rats. Means k SE of data from 5 rats. Heavy horizontaZ bar represents period of feeding and darkness (1800 - 0600). Lines of best fit of total plasma calcium (Ca) against time (t ) in days, and correlation coefficient (r), for each selected time are: Ca (0600) = 8.56 - 0.204 t, r = 0.59; Ca (1000) = 9.34 - 0.158 t, r = 0.63; Ca (1400) = 9.81 - 0.177 t, r = 0.48; Ca (1800) = 9.43 - 0.177 t, r = 0.74; Ca (2200) = 8.23 - 0.204 t, r = 0.64; Ca (0200) = 8.51 - 0.362 t, r = 0.67. These correlations are significant at P < 0.001. Mean 24-h values t, SE of total plasma calcium on the lst, 2nd, 4th, 6th, and 8th experimental days are shown at bottom. FIG.
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (139.184.014.150) on August 11, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
CIRCADIAN
RHYTHMS
IN
CALCIUM
R313
METABOLISM
crease to 7.42 t 0.19 mg/lOO ml 1 wk later. In control rats, as expected, the mean value remained unchanged (Fig. 1). In both control and calcium-deficient rats, significant daily variations in total plasma calcium were observed. In control rats, the maximum amplitude of the daily variation was around 0.65 mg/lOO ml, or 6% per day. The rats subjected to a calcium-deficient regimen developed progressively greater fluctuations (Fig. 2). Thus, on the 1st experimental day, total plasma calcium variation was 1.2 mg/lOO ml (or 12.5%), whereas 1 wk later, it was 2.5 mg/lOO ml (or 30%), falling from 8.57 t 0.41 mg/lOO ml at 1400 to 6.23 t 0.42 mg/lOO ml at 0200. As the values observed during the fluctuations in total plasma calcium of control rats were similar from 1 day to another, the data between days were pooled (Fig. 1). The temporal characteristics of daily total plasma calcium fluctuations were as follows: from the nadir at 2200 the levels rose steadily to reach a maximum at 1400. Thereafter, the levels fell steadily during the next 8 h. The preprandial fall was significant (P = 0.002). Although the pattern of the daily fluctuation was not greatly affected by feeding a calcium-deficient regimen, the level of the mean plasma calcium fell. As in the control group, the biphasic shape of daily fluctuations was centered around 1400 (i.e., 4 h before feeding). Plasma calcium levels decreased more sharply than in controls, reaching progressively lower values on each day. Thus, the mean plasma calcium level measured at the same time each day decreased as a linear function of the duration of the calcium deficiency at a rate of about 0.2 mg/lOO ml per day with the exception of that at 0200 when it decreased twice as rapidly. The lines of best fit are defined by the equations reported in the legend of Fig. 2. The daily fluctuations of both plasma calcium and magnesium levels in control and calcium-deficient rats (e;rpt B) are shown in Table 1. After 18 days of calcium deficiency, the mean 24-h value of total plasma magnesium was significantly increased (P < 0.001). In calciumdeficient rats, the total plasma calcium and magnesium TABLE
control
1. Total plasma calcium and magnesium and calcium-deficient rats Calcium,
Time
of Day,
1800 2200 0200
1000 1400
24-h
mean
h
mg/lOO
ml
Control
Calcium-deficient
10.84 kO.10 10.32 kO.08 10.47 kO.08 10.70 kO.08 10.91 kO.07 11.22 kO.07 10.74 kO.05
9.87 kO.18t 7.77 *0.53 * 7.99 t0.28* 8.15 *0.33* a.90 *0.28* 8.95 *o. 13* 8.61 kO.16*
Values are means t, SE for 5 rats. Food dark period (1800-0600). * P < 0.001; 0.05; 5 NS for control vs. calcium-deficient
Magnesium,
1.63 *0.04 1.68 t0.04 1.69 t0.04 1.68
zto.03 1.65 *0.02 1.63 to.03 1.66 kO.014 tP
rats.
available
STAUB, MILHAUD
Service de Biophysique,
ANNE-MARIE
CHU Saint-Antoine,
ANNE-MARIE PERAULT-STAUB, STAUB, JEAN-FRAN~OIS, AND GERARD MILHAUD. Endogenous nature of circadian rhythms in calcium metabolism. Am. J. Physiol. 237(5): R3llR31’7, 1979 or Am. J. Physiol.: Regulatory Integrative Comp. Physiol. 6(3): R311-R317, 1979.-Rats studied when the lights are on from 0600 to 1800 daily and fed only in the dark period displayed circadian rhythms in plasma calcium (ionized and total) and 45Ca concentrations, 6 and 8 days after 45Ca administration. In rats fed a calcium-deficient diet, the amplitude of daily variation of plasma ionized and total calcium increased remained esmarkedly whereas plasma 45Ca daily fluctuation sentially unchanged. In the calcium-deficient rats, significant correlations between plasma calcium and 45Ca and between plasma calcium and magnesium were observed throughout the 24 h; circadian periodicity of calcium metabolism persisted in rats fasted overnight, regardless of the illumination schedule. Normal daily fluctuations in plasma 45Ca, lost after thyroparathyroidectomy (TPTX), were restored by feeding the TPTX rats a high-calcium diet. These results demonstrate clearly that circadian rhythms of calcium metabolism occurred irrespective of the light-dark schedule, the calcium supply through intestines and the thyroparathyroid system. An attractive suggestion is that circadian rhythmicity originates as a result of dynamic properties involving nonlinear processes of calcium metabolism. rat; calcium
homeostasis;
dissipative
structure
INVESTIGATORS have demonstrated the existence of circadian rhythms that regulate calcium metabolism in the rat. These studies include observations on the circadian rhythms in total (7, 19) and ionized plasma calcium (4)) urinary calcium excretion (2)) serum calcitonin (M), and metabolic activity of bone (16). In some experiments in rats, the patterns of the daily fluctuations both in plasma calcium and its radionuclide concentration resulting from the administration of 45Ca 1 wk before the experiments were measured (11, 19). All of these data, favoring the existence of “internal periodic changes” in calcium homeostatic processes that could be correlated with the periodic environmental events, led us to postulate a new concept for calcium homeostasis (12), namely that rhythmicity must be a fundamental requisite for normal calcium metabolism. The aim of the present investigation was to obtain further insight into the origin and the role of the internal circadian processes in calcium regulation. The daily fluctuations in plasma calcium and 45Ca concentrations were used as measures of the calcium metabolism processes in rats fed an essentially calcium-free diet where the usual
MANY
0363~6119/79/~-~$01.25
Copyright
0 1979 the American Physiological
rhythms
PERAULT-STAUB, 75571 Paris Ce’dex 12, France
periodic calcium supply through the intestinal absorption is absent. This study also included the effect on circadian rhythms of a 24-h fast either in the dark or in the usual light-dark schedule, and of thyroparathyroidectomy with or without access to a high-calcium diet. The results in this report emphasize the need to consider rhythmicity as a primordial endogenous characteristic of the physiological control of calcium metabolism; the results also help clarify the role of these endogenous regulatory processes in calcium homeostasis. METHODS
Animals Weanhng male Wistar C.F. rats were placed in special cages adapted for automatic control of lighting and access to food. The rats were exposed to darkness (1800-0600) on a 12-h light-dark (LD) schedule. Food was available only during the dark period; water was provided ad libitum throughout the 24 h.
Diets The semisynthetic diet (U.A.R., Villemoisson/orge, France) contained 0.7% phosphorus; calcium carbonate was added to reach the desired level. The calcium-deficient regimen was essentially free of calcium (less than 0.03%). The normal diet contained 0.7% calcium and the high-calcium diet, 1.5% calcium. The diets contained adequate amounts of vitamin D3 and necessary minerals.
Protocols Effect of calcium-deficiency. EXPERIMENT A. During the 1st postweanhng wk, 80 animals were fed a normal diet and drank tap water; they were then divided into two equal groups. During the next 3 wk, the experimental group was restricted to a calcium-deficient diet and distilled water, and the control group was fed a normal diet and tap water. Two weeks later the rats were weighed, individually stamped, randomly allocated into subgroups of five rats each, and injected intravenously with radioactive calcium (100 PCi of 45Ca as CaC12). At selected times during the day (0600, 1000, 1400, 1800, 2200, or 0200) rats in the subgroups were weighed and blood samples (0.1 ml) were obtained by retro-orbital puncture. The blood was collected in heparinized tubes and centrifuged immediately. On the 8th day, a larger blood sample (1.5 ml) was obtained by cardiac puncture in a lightly Society
It311
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (139.184.014.150) on August 11, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
R312
STAUB,
heparinized syringe; the syringe was centrifuged immediately, the piston down. An aliquot of the plasma sample was used immediately to measure ionized calcium, the remainder was used to determine total calcium. EXPERIMENT B. The effect of calcium deficiency on plasma magnesium was studied in a separate experiment. The experimental group had access to a calcium-deficient diet and distilled water for 18 days; the rats in the control and experimental groups were bled by retro-orbital puncture only once during one 24-h period. Effect of fasting. Weanling rats were fed a normal diet and received tap water ad libitum for 3 wk; they had access to food only during the dark period on a 12-h LD schedule. Radioactive calcium (100 &i of 45Ca as CaC12) was administered 1 wk before the first blood sample was obtained. At 1800 on the experimental day, one group was allowed the usual access to food and the 12-h LD schedule; for the two other groups, food and the sawdust litter were removed, distilled water was available and the illumination schedule was either unchanged (second group) or replaced by constant darkness (third group). Rats, 5 to 10 per subgroup, were bled only once by retroorbital puncture at 4-h intervals during any one 24-h period. Effect of thyroparathyroidectomy. Parathyroidectomy was performed by blunt dissection under ether anesthesia on 2-mo-old rats. (Animals were considered successfully parathyroidectomized if the plasma calcium was less than 8.0 mg/lOO ml 2 days after the operation.) Five days later, the animals previously maintained on the regular diet were distributed into two groups of 40 rats each. One group was maintained on the normal diet, the other was placed on a high-calcium diet. The radioactive calcium was administered (100 @i of 45Ca as CaC12). Five days later, the rats were thyroidectomized. Experimental blood samples were collected 2 days after thyroidectomy, at a time when the deprivation of endogenous iodinated hormones was not apparent. Thyroparathyroidectomized rats (TPTX) six to seven per subgroup, were bled by retro-orbital puncture at 4-h intervals; the rats were only bled one time during a 24-h period.
PERAULT-STAUB,
AND
MILHAUD
RESULTS
Total Plasma Calcium and Magnesium Control and Calcium-Deficient Rats
in
Figures 1 and 2 show the levels of the total plasma calcium in control and calcium-deficient rats, (expt A). Three factors were considered: 1) the mean 24-h value, 2) the amplitude of the daily variation, and 3) the pattern of the daily fluctuation. On the 1st day of the experiment (after 2 wk on the calcium-deficient diet) the mean 24-h plasma calcium had fallen to the level of 9.01 t 0.14 mg/lOO ml (Fig. 2) compared to 10.99 t 0.07 mg/lOO ml in control rats (Fig. 1). The difference was highly significant (P < 0.001). The mean value in calcium-deficient rats continued to de-
mean zlh-vsluo day O-l - l-2 - 3-4 - 5-6 (SO) ” 7-0
(341 (341
(60)
(50)
(mg/lOOml) 10.99 +0.07 11.22 to.07 11.07 zo.04 11.10 r0.09 11.14 TO.06
(32)
t (3s)
FIG. 1. Circadian variation in total plasma calcium of control rats. Pattern of 24-h variation was highly reproducible from 1 day to the next. Data, means & SE, were pooled over the &day period. Numbers in parentheses indicate number of samples used for estimation of mean 5-day value at each selected time. Heavy horizontaz bar represents time of feeding and darkness. Preprandial fall between 1400 and 1800 was significant, P = 0.002. Mean 24-h values of total plasma calcium on each of 5 days of experimental week are listed on right.
Blood Analysis
Plasma total calcium and magnesium were determined by atomic absorption spectrophotometry (Perkin-Elmer model 503). Plasma ionized calcium concentration was measured anaerobically with model SS-20 ionized calcium analyzer (Orion Research, Cambridge, MA). Plasma radioactivity was measured in a liquid scintillation counter, corrected for the daily body weight to eliminate individual fluctuations and expressed as dpm/ml. moan 24hualu:
Statistical
Treatment
Rats were assigned randomly into experimental and treatment groups. The data from each experiment were subjected to analysis of variance, and standard error of the means (SE) were calculated. Statistical analysis between groups was performed by Student’s t test; P < 0.05 was considered as significant. Correlation coefficients (r ) between parameters were calculated from the individual values bv the method of linear least sauares.
0 9.Ol to.14 8.63tO.14
8.43zO.13
7.98?0.14
7.42:0.19(mg /lOOml )
2. Circadian variation of total plasma calcium in calcium-deficient rats. Means k SE of data from 5 rats. Heavy horizontaZ bar represents period of feeding and darkness (1800 - 0600). Lines of best fit of total plasma calcium (Ca) against time (t ) in days, and correlation coefficient (r), for each selected time are: Ca (0600) = 8.56 - 0.204 t, r = 0.59; Ca (1000) = 9.34 - 0.158 t, r = 0.63; Ca (1400) = 9.81 - 0.177 t, r = 0.48; Ca (1800) = 9.43 - 0.177 t, r = 0.74; Ca (2200) = 8.23 - 0.204 t, r = 0.64; Ca (0200) = 8.51 - 0.362 t, r = 0.67. These correlations are significant at P < 0.001. Mean 24-h values t, SE of total plasma calcium on the lst, 2nd, 4th, 6th, and 8th experimental days are shown at bottom. FIG.
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (139.184.014.150) on August 11, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
CIRCADIAN
RHYTHMS
IN
CALCIUM
R313
METABOLISM
crease to 7.42 t 0.19 mg/lOO ml 1 wk later. In control rats, as expected, the mean value remained unchanged (Fig. 1). In both control and calcium-deficient rats, significant daily variations in total plasma calcium were observed. In control rats, the maximum amplitude of the daily variation was around 0.65 mg/lOO ml, or 6% per day. The rats subjected to a calcium-deficient regimen developed progressively greater fluctuations (Fig. 2). Thus, on the 1st experimental day, total plasma calcium variation was 1.2 mg/lOO ml (or 12.5%), whereas 1 wk later, it was 2.5 mg/lOO ml (or 30%), falling from 8.57 t 0.41 mg/lOO ml at 1400 to 6.23 t 0.42 mg/lOO ml at 0200. As the values observed during the fluctuations in total plasma calcium of control rats were similar from 1 day to another, the data between days were pooled (Fig. 1). The temporal characteristics of daily total plasma calcium fluctuations were as follows: from the nadir at 2200 the levels rose steadily to reach a maximum at 1400. Thereafter, the levels fell steadily during the next 8 h. The preprandial fall was significant (P = 0.002). Although the pattern of the daily fluctuation was not greatly affected by feeding a calcium-deficient regimen, the level of the mean plasma calcium fell. As in the control group, the biphasic shape of daily fluctuations was centered around 1400 (i.e., 4 h before feeding). Plasma calcium levels decreased more sharply than in controls, reaching progressively lower values on each day. Thus, the mean plasma calcium level measured at the same time each day decreased as a linear function of the duration of the calcium deficiency at a rate of about 0.2 mg/lOO ml per day with the exception of that at 0200 when it decreased twice as rapidly. The lines of best fit are defined by the equations reported in the legend of Fig. 2. The daily fluctuations of both plasma calcium and magnesium levels in control and calcium-deficient rats (e;rpt B) are shown in Table 1. After 18 days of calcium deficiency, the mean 24-h value of total plasma magnesium was significantly increased (P < 0.001). In calciumdeficient rats, the total plasma calcium and magnesium TABLE
control
1. Total plasma calcium and magnesium and calcium-deficient rats Calcium,
Time
of Day,
1800 2200 0200
1000 1400
24-h
mean
h
mg/lOO
ml
Control
Calcium-deficient
10.84 kO.10 10.32 kO.08 10.47 kO.08 10.70 kO.08 10.91 kO.07 11.22 kO.07 10.74 kO.05
9.87 kO.18t 7.77 *0.53 * 7.99 t0.28* 8.15 *0.33* a.90 *0.28* 8.95 *o. 13* 8.61 kO.16*
Values are means t, SE for 5 rats. Food dark period (1800-0600). * P < 0.001; 0.05; 5 NS for control vs. calcium-deficient
Magnesium,
1.63 *0.04 1.68 t0.04 1.69 t0.04 1.68
zto.03 1.65 *0.02 1.63 to.03 1.66 kO.014 tP
rats.
available
