The interaction of lithium and time-of-day on calcium, magnesium, parathyroid hormone, and calcitonin in rats

/??yc,hiarr.v Research,

7, I2 I- 13 I

( 1980)

121

Elsevier Biomedical Press

The Interaction Magnesium, Rats

of Lithium Parathyroid

and Time-of-Day Hormone, and

Donald L. McEachron, Daniel F. Kripke, Dennis Pavlinac, and Leonard Deftos

Maryruth

on Calcium, Calcitonin in

Eaves, Linda Lenhard,

Received December 2, 1981; revised version received March 19, 1982; accepted March 29, 1982. Abstract. Effects of lithium on the concentrations and temporal patterns of serum and cerebellar calcium and magnesium, parathyroid hormone, and calcitonin were studied in 186 rats sacrificed around 24 hours of clock time. Serum calcium, serum and cerebellar magnesium, and parathyroid hormone were increased and calcitonin decreased in lithium-fed animals. Lithium-fed rats also showed different temporal patterns in serum calcium, parathyroid hormone, cerebellar magnesium, and calcitonin. Data support the hypothesis that lithium competes for calcium receptor sites, causing a compensatory increase in parathyroid hormone and decrease in calcitonin until a new, higher set-point for calcium is established. Lithium strongly affected biological rhythms, an effect which may account in part for the diverse tion.

literature

Key Words. Lithium,

on lithium’s

influence

mineral regulation,

on calcium

and

magnesium

regula-

calcium, magnesium,

circadian

rhythms.

is a lack of clear consensus on how lithium acts on mineral metabolism. Lithium treatment in animals or humans has been variously reported as increasing serum calcium (Andreoli et al., 1972; Mellerup et al., 1973; Christiansen et al., 1975; Mellerup et al., 1976; Thysell et al., 198 I), decreasing serum calcium (Tupin et al., 1968), or having no effect (Frizel et al., 1969; Plenge and Mellerup, 1976). Similarly, one study reported that lithium treatment decreased serum magnesium (Frizel et al., 1969), others described significant increases (Andreoli et al., 1972; Birch and Jenner, 1973; Christiansen et al., 1975; Transbsl et al., 1978; Thysell et al., 1981), and still others found no lithium-associated change in serum magnesium (Dunner et al., 1975; Pederson et al., 1977). Lithium was reported to increase magnesium in the brain by King et al. (1969) and Essman (1975) and to decrease magnesium in the brain by Birch and Jenner (1973) and Bond et al. (1975). There are a number of possible explanations for these conflicting results. Differences in the route of administration and differences in the duration of treatment may be important. One possibility that has not yet been adequately examined involves lithium’s effect on biological rhythms. There

Donald L. McEachron, B.A., is at Department of Neurosciences,

School of Medicine; Daniel F. Kripke, M.D., and Dennis Pavlinac, M.D., are at Department of Psychiatry; and Leonard Deftos, M.D., is at Department of Medicine, University of California, San Diego. Maryruth Eaves and Linda Lenhard are at San Diego Veterans Administration Medical Center, as are McEachron, Kripke, Pavlinac, and Deftos. (Reprint requests to Dr. D.F. Kripke, Dept. of Psychiatry (I 16A), VAMC, 3350 La Jolla Village Dr., San Diego, CA 92161, U.S.A.) 0165-1781;82/0000-0000/$02.75

0 Elsevier Biomedical

Press

122 Lithium’s action on serum calcium varied with the time of sampling according to Mellerup et al. (1976); however, the statistic used to analyze the data (Student’s t test) involved multiple testing, and no conclusion concerning the overall effect of lithium therapy was possible. Twenty-four hour rhythms have been described for serum magnesium (Touitou et al., 1978) parathyroid hormone (PTH), and serum calcium (Jubiz et al., 1972; Lo Cascio et al., 1977). Lithium has been conclusively demonstrated to lengthen or delay various overt circadian rhythms in animals (Hofman et al., 1978; Kripke and Wyborney, 1980; McEachron et al., 1981) and humans (Kripke et al., 1979; Johnsson and Engelmann, 1980). In a previous publication, we demonstrated that lithium treatment had various effects on biological rhythms, including the rhythms of calcium magnesium, PTH, and calcitonin. These effects included phase delays and altered wave forms (McEachron et al., 1982). The time of sampling, combined with lithium’s effects on biological rhythms, could account for much disparity among results (see Fig. 1).

Fig. 1. Hypothetical

effect of biological

rhythm phase

1

I

1

I

6

10

14

18

I

22

I

I

2

6

TIME If the only effect were to shift the phase of a substance’s biological rhythm, significant increasesand decreases In the concentration of that same substance might be found by sampling at different times of the day.

Lithium’s effect on circadian rhythms might underlie its therapeutic efficacy or it might be incidental. Effects on the mean concentration of minerals and related hormones could be an alternative mode of therapeutic action. It is important, therefore, to present the effects of lithium on mineral regulation, both to provide clues as to

123 lithium’s mode of action and to understand complications arising from long-term lithium therapy. This presentation emphasizes the effects on mineral regulation in rats given several weeks of lithium treatment. Blood concentrations of calcium, magnesium, calcitonin (CT), PTH, and lithium, and cerebellar levels of calcium, magnesium, and lithium were sampled at regular intervals around the clock to examine the changes, if any, caused by lithium treatment, while controlling for lithium’s known chronobiological effects.

Methods Procedure. The procedure used in this experiment is described in greater detail in our previous publication (McEachron et al., 1982). One hundred and eighty-six male Sprague-Dawley rats (Hilltop) were studied in July and August, 1979. Ninety-one were randomly selected as controls and received Rodent Laboratory Chow 5001 ad lib. The remaining 95 animals received Rodent Chow 5001 with 0.3% lithium carbonate (300 mg lithium carbonate/ 100 g chow) and were maintained on this diet for at least 3 weeks. The animals were exposed to a light/dark cycle (12.5: 11.5) with light on from 0500 to 1730 hours. Animals were sacrificed in &hour sessions which covered 24 hours of clock time over 3 days. Six rats, three lithium-fed and three control, were sacrificed every 2 hours in each 8-hour session. The first sessions were from 2100 to 0500,050O to 1300, and 1300 to 2100 hours, and 2 days later, the procedure was repeated with sessions from 0100 to 0900,090O to 1700, and 1700 to 0100 hours. Within each 2-hour interval, the sacrifice procedure was alternated between lithium-fed and control animals and this order was alternated between consecutive 2-hour intervals. Near the time when the lights were switched on or off (0400 to 0600 and 1630 to 1830 hours), I7 animals were sacrificed, 8 before the change and 9 afterwards. The room where the animals were sacrificed was well-lit (700 lux) from 0500 to 1730 hours. From 1730 to 0500 hours, it was lit by two 7.5 watt red photographic safelights, each at least 3 feet from the animals at all times. Blood was withdrawn by cardiac puncture after the rats were anesthetized by an intraperitoneal injection of sodium pentobarbital(8.75 mg/ 100 g body weight). Blood samples were spun for IO minutes in a refrigerated centrifuge. Serum and plasma were frozen for later analyses. Seven of the 17 animals sacrificed near the lighting changes were killed by CO, inhalation, and no blood was obtained from these animals. Within 2 minutes after bleeding or CO, exposure, the cerebellum and liver of each animal were removed and frozen on solid CO,. These samples were stored at -27°C for later analysis. Biochemical

Analysis.

The biochemical

methods

used are described

in McEachron

et al.

(1982). Analysis. The data were analyzed using three-way analyses of variance(ANOVA). The main effects were lithium-fed vs. control, time-of-sampling, and first vs. second replication. The important contrasts examined by ANOVA were: (I) the main treatment effect indicating the overall effect of lithium on concentration; (2) the time-treatment interaction effect, which indicated whether lithium had altered the 24-hour rhythm in concentrations of the substances; and (3) the replication-treatment interaction effect, which showed whether lithium’s effect was stable between replications. Instability of lithium effects between replications could result either from the longer duration of treatment received by the animals sacrificed in the second replication or from factors related to the timing of the sacrifice sessions. For each variable and effect, the F max test for heterogeneity of variances was performed, and if significant differences in the variances were found (a violation of one of the assumptions of ANOVA), the variable was converted into base IO logarithms (Sokal and Rohlf, 1969). Correlations were calculated for all animals of both replications taken together and also for each of the six 8-hour sampling sessions taken separately in order to control for the possible effects of rhythmicity and replication.

Statistical

124 Results Significant increases occurred in the lithium-fed rats in concentrations of serum calcium, serum magnesium, cerebellar magnesium, and plasma PTH. Plasma CT was significantly decreased. Cerebellar calcium was not significantly altered. Four of the six substances examined showed significant time-treatment interactions: serum calcium (F = I .905, LIP= 1 I, I I 1, p < 0.05) cerebellar magnesium (F = 14.670, df= 1 I, 116,p<0.001),CT(F=3.208,~~=11,110,p<0.01),andPTH(F=5.582,df=11, 10 1, p < 0.00 1). Serum magnesium just barely missed a significant interaction effect at the 0.05 level (F= 1.830, gf= 11, 1 IO, p < 0.06). For cerebellar calcium the difference in temporal pattern between the lithium-fed and control groups was not significant, perhaps because of large interanimal variability. (See McEachron et al., 1982, for further discussion of lithium’s effect upon these and other rhythms.) Table 1 presents the main results of this study. Calcitonin and PTH results had variances sufficiently heterogeneous to require conversion to logarithms for the ANOVA. With the sole exception of serum magnesium, none of the substances examined showed significant replication-treatment interaction effects or significant differences in either control or lithium-fed groups between the first and second replications.

Table 1. Comparison rats

of the overall concentrations

of control and lithium-fed

Substance (n)

Mean (k SD)

Lithium treatment

Serum calcium Control (80 I Lithium (79)

10.16 mg/dl 10.52 mg/dl

I? 0.631 ct 0.71 i

i= = 12.839, df = 1, 111,

Cerebellar Control Lithium

calcium (731 (891

9.27 mg/dl 10.23 mg/dl

I? 4.61) (l 8.771

/= = 0.789, df = 1, 114, p < 0.50

Serum magnesium Control (80) Lithium 1781

2.20 mg/dl 2.48 mg/dl

12 0.451 (2 0.44)

F= 19.098, df=

effect

p < 0.001

1, llO,p’O.OOl

Cerebellar Control Lithium

magnesium (74 I (90)

14.38 mg/dl 14.91 mg/dl

12 1.121 I+ 1.33)

/= = 8.413, df = 1, 116,

Calcitonin Control Lithium

(log) (791 1791

70.48 pg/ml 56.12 pg/ml

(k 35.90) (i 22.481

,==18.285,df=1,110,p~0.001

PTH Control Lithium

(621 1701

189.55 pg/ml 235.39 pg/ml

(? 134.88) ik 151.101

Liver glycogen Control 1891 Lithium (901

34.94 mcq/mg 24.44 mcq/mg

Lithium concentrations

Mean k SEM

Serum 1721 Cerebellar (90

1.34 mEq/l 1.64 mEq/l

1

(2 14.241 I+ 12.71 I

(2 0.25 (2 0.47

1 1

F= 12.287, df=

p < 0.01

1, lOl,p
F=31.581,df=1,131,p~0.001

Paired t test t = 4.41 Ip < 0.001,

125 There was a significant replication-treatment interaction for serum magnesium (F= 6.321, df= I, 110, p < 0.05). Separate ANOVAs run on the lithium-fed and control groups indicated that the control group was stable at about 2.20 mg/ dl (F= 0.064, df = I, 78, NS). In the lithium-fed group, the mean serum magnesium level fell from 2.66 mg/dl ? 0.44 (SD) in the first replication to 2.30 mg/dl ? 0.36 (SD) in the second replication (F = 13.96, df = 1, 76, p < 0.001). Mean serum lithium levels were stable over the two replications, although there was a significant effect due to time-of-sampling (F = 2.257, df = 11, 48, p < 0.05). Cerebellar lithium concentrations were significantly higher than those in serum (see Table 1). There was a significant replication effect for cerebellar lithium. The mean concentration of cerebellar lithium rose from 1.53 mEq/ 1 * 0.47 (SD) in the first replication to 1.75 mEq/ I IL 0.49 (SD) in the second replication. The effect of time-of-sampling was not significant for cerebellar lithium. Table 2 displays the correlation matrices for serum calcium and magnesium, PTH, and CT, comparing the control and lithium-fed groups. The first correlations were created from all data, with log values for CT and PTH. The correlations in parentheses are averages of six correlations, one for each g-hour session, using raw data. When considered in separate g-hour bins, none of the variances were sufficiently heterogeneous to require conversion to logarithms. Almost all correlations were smaller for the lithium-fed group. When the matrices, using all the data taken together, were converted to covariance matrices and compared (Morrison, 1976), the intercorrelations of the lithium-fed animals were found to be significantly smaller than those of controls (~2 = 343, df= 10, p < 0.005). Table 2. Correlation

matrices for control and lithium-fed

animals

Control animals

S-Ca

S-Ca

S-Mg

CT (log)

PTH (log)

1.0

0.623t r0.629ti

0.309t (0.321t)

0.547t (0.350t)

1.0

0.476t (0.503t)

0.243* 10.288'1

1.0

0.390t 10.479ti

S-Mg CT (log) PTH (logi Lithium

S-Ca

S-Mg CT

1 .o animals

S-Ca

S-Mg

CT (log)

PTH (log)

1.0

0.447t (0.578t)

0.145 10.084)

0.176 (0.1151

1.0

0.263* 10.1021

0.204 (0.316t)

1.0

0.043 (0.0371

i log I

1.0

PTH (loq) * = p < 0.05; f =p < 0.01; unmarked = not significant. Correlations in parentheses are averages of the B-hour sampling correlations are from all data considered as a whole.

sessions,

considered

separately.

The other

There were no significant correlations among any of the variables and cerebellar calcium or magnesium when all data were considered together. When the data were

126

analyzed in S-hour bins and then averaged, significant correlations appeared in the control group between CT and cerebellar calcium (r = -0.264, p < 0.05). Serum lithium showed significant correlations only when the data were correlated in 8-hour bins. Serum lithium was significantly correlated with serum calcium (r = 0.295, p < 0.05) and with serum magnesium (Y = 0.245, p < 0.05). When all data were taken together, cerebellar lithium was correlated with body weight (r = -0.270, p < 0.05). Previous experiments with this lithium diet have shown that it causes significantly more drinking, increasing intake from 42 ml/ day f 7.2 (SD) in controls to 252 ml/ day ? 57.2 (SD) in lithium-fed rats. At the end of the experiment, control animals weighed an average of 492 g ? 3.5 (SEM), and lithium-fed animals had a mean weight of 358 g + 2.8 (SEM). Although the animals had weighed 400 ? IO (SD) g at the start of the experiment, the only significant correlations observed for body weight were with cerebellar lithium (see above) and with PTH in the lithium-fed group (r = -0.35 I, p < 0.0 I ). Both lithium-fed and control groups weighed more at the time of the second replication, controls gaining an average of 0.7% of body weight with reference to the first replication and lithium-fed animals gaining 0.9%. The rhythm in liver glycogen had no significant time-treatment interaction effect (F = 1.195, @‘= 1I, 131, NS). Discussion As reflected in Table 1, lithium’s effects on serum calcium (S-Ca) and magnesium (S-Mg), cerebellar magnesium (C-Mg), CT, and PTH concentrations were all highly significant, and with the exception of S-Mg, independent of replication effects. Lithium treatment caused significant increases in the levels of S-Ca, S-Mg, and PTH, while causing a significant decrease in CT. Cerebellar calcium (C-Ca) was not significantly altered. These overall results are in agreement with some recent reports (King et al., 1969; Mellerup et al., 1973; Christiansen et al., 1975; Essman, 1975; Thysell et al., 198 I) and contrast with others (Tupin et al., 1968; Frizel et al., 1969; Birch and Jenner, 1973; Bond et al., 1975). The figures and the time-treatment interactions results help explain the disparities in previous results. Consider serum magnesium, for example (Fig. 2~). Clearly, if each time point were sampled separately and a t test applied, different results would be found for serum magnesium at 1000 and 2200 hours or in PTH at 1000 and 2200 hours (Fig. 2h). It should be emphasized that simply putting control and lithium-fed animals on the same light-dark schedule and sacrificing at the same time does not adequately control effects of lithium on biological rhythms. One-time sampling is simply not sufficient to study biochemical effects of lithium treatment. The diet was chosen as the preferred route of lithium administration to avoid exposing treated animals to any additional time cues not available to the controls. For example, hypertonic intraperitoneal lithium injections cause extensive tissue damage and result in considerable physical discomfort not caused by similar injections of saline (Smith, 1976). Thus, the time cues given by injections in the two groups would not be equivalent. Lithium salts cause flavor aversion in rats (Smith, 1977), resulting in some weight loss in lithium-fed animals. This problem could not be corrected by

127

Fig. 2. Concentrations in serum magnesium (a) and plasma PTH (b) forcontrol and lithium-fed rats over 24 hours of clock time

2.9

q

Control

0

Lithium-fed

2.8 2.7 _

2.6

?

2.5

F

H 1

2.4 2.3

z 5

2.2

z

2.1 2.0 i .9 1.8 1.7 1400

TIME

(Hour)

2.7-

q

Control

0

Lithium-fed

2.6 2.5 2.4 z \ E z z Q_

2.3~ 2.2

2.0 1 .9 1.8 1.7 1.6

-

2.1 1.5 I 0600

1800

TIME The results shown are the means of the two replications.

(Hour

Vertical

1

bars represent

+ 1 SEM

128 pair-feeding insofar as that would involve restricting the access to food for the control animals, which is itself a potent time cue (Krieger, 1974; Nelson et al., 1975), then available only to controls. Salt-fed (NaCl) animals might be a useful control group, but salt also affects calcium homeostasis (Goulding, 1980). It is possible that flavor aversion was a factor in lithium effects on substance concentrations, but weight loss probably does not explain effects of lithium on biological rhythms, since the rhythm in liver glucogen, one of the most sensitive to feeding effects (Nelson et al., 1975; Martin et al., 1979) was not altered.

Fig. 3. Concentrations in cerebellar lithium (higher unbroken line) and serum lithium (lower dotted line) over 24 hours of clock time

2.0 1.9 1.8 1.7 t

1.6

Iz E

1.5 1.4

H 1 I

1.3

tz

1.2 1.1 1.0 0.9

-

LIGHTS ON

OFF

0.8 9 2

6

10

14

18

22

2

6

TIME (Hour) The results shown are the means of the two replications

Vertical

bars represent

2 1 SEM

Considering the data as a whole, we are faced with an interesting lithium-fed rats were hypercalcemic. but CT and PTH were responding

paradox. The as if they were

129 hypocalcemic. Given lithium’s chemical similarities to calcium and magnesium (Birch, 1970), these results suggest that lithium was acting as a false transmitter, binding to calcium receptor sites in PTH- and CT-secreting cells and causing them to underestimate the amount of available calcium. The resulting increase in PTH and decrease in CT would create higher concentrations of blood calcium and magnesium. Brown (1980) recently examined the effects of lithium chloride on cultured bovine parathyroid cells. After the cells were incubated with various concentrations of lithium chloride (LiCl), it was found that as the concentration of LiCl was increased, the concentration of calcium needed to inhibit PTH secretion increased also. The effect, requiring preincubation with LiCl and persisting after the LiCl was washed out of the surrounding medium, led Brown to suggest that lithium was affecting some intracellular process. Lithium’s effect in vivo on PTH- and CT-secreting cells could be similar to its effect on parathyroid cells in vitro. In effect, lithium would cause these cells to underestimate blood calcium concentrations, creating a higher set-point for calcium. How are the changes in S-Mg and C-Mg brought about? S-Mg can be increased by lithium independently of the thyroid and parathyroid glands insofar as these glands can be surgically removed without altering the lithium-induced increase in S-Mg (Plenge and Mellerup, 1976). What caused the increases in S-Mg and C-Mg is unknown, but may involve competition for the synthesis of bone material (Plenge and Mellerup, 1976) or lithium-induced alterations in carbohydrate metabolism (Mellerup et al., 1973). Combined with the significant replication effect in S-Mg, these data suggest that the changes caused by lithium in S-Ca and S-Mg may have separate origins. Clinically, this work suggests that the increases in S-Mg and S-Ca often seen within lithium treatment are expected side effects which do not require their own treatment. However, the long-term effects of the disruption of calcium and magnesium regulation are not known and should be investigated to minimize complications of long-term lithium therapy. Acknowledgment. We wish to thank Dr. Robert Elmasian, Dr. Erhard Haus, Paul Clopton, lngrid Lewin, Frank Taylor, and the members of the San Diego Veterans Administration Medical Center Animal Research Facility for their advice and technical assistance. This work was supported in part by the Medical Research Service of the Veterans Administration, the American Cancer Society, grants AM- 15888 and AM-25604 from the National Institutes of Health, and by an RSDA, 5 K02 MH-00117 from the National Institute of Mental Health. References Andreoli, V.M., Villani, F., and Brambilla, G. Increased calcium and magnesium excretion induced by lithium carbonate. P.s_~~c,hopharmac,ologia, 25, 77 (1972). Birch, N.J. Lithium and plasma magnesium. British Journal of’ Px~~dziatr~s, 116, 461 (1970). Birch, N.J.. and Jenner, F.A. The distribution of lithium and its effects on the distribution of other ions in the rat. British Journal ctf Pharmacolog,~. 47, 586 (1973). Bond, P.A.. Brooks, B.A., and Judd, R. The distribution of lithium, sodium, and magnesium in rat brain and plasma after various periods of administration of lithium in the diet. British Journal

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130

Brown, E.M. Lithium induces abnormal calcium-regulated PTH release in dispersed bovine parathyroid cells. Clirzic~l Research. 28, 646A (1980). Christiansen, C., Baastrup, P.C., and Transbel, I. Osteopenia and dysregulation of divalent cations in lithium-treated patients. Neurol’.~.,,c,hohio/og~, 1, 344 ( 1975). Dunner, D.L., Meltzer, H.L., Schreiner, H.C., and Feigelson, J.L. Plasma and erythrocyte magnesium levels in patients with primary affective disorder during chronic lithium treatment. Actu fswhiarrica Scandinuvica, 51, 104 (1975). Essman, W.B. Lithium. Lancet, II, 547 (1975). Frizel, D.A., Coppen, A., and Marks, V. Plasma magnesium and calcium in depression. British Journal of Ps.whiatry, 115, I375 ( 1969). Goulding, A. Effects of dietary NaCl supplements on parathyroid function, bone turnover and bone composition in rats taking restricted amounts of calcium. Mineral Electr(l/J,te Meraho/ism, 4, 203 ( 1980). Hofmann, K., Gunderott-Palmowski, M., Wiedermann, G., and Engelmann, W. Further evidence for period lengthening effect of Li’ on circadian rhythms, ZeirschrlfiJtir Nururjkwhung,

Johnsson,

33, 23

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effects on electrolyte

excretion.