IS THE BLOOD CALCIUM LEVEL OF MAMMALS INFLUENCED BY ESTROGENIC SUBSTANCES?1 LOUIS LEVIN2 AND PHILIP E. SMITH From the Department of Anatomy, College of Physicians and Surgeons, Columbia University N E W YORK CITY
The literature on the relationship of the gonads to the level of calcium in the blood has recently been reviewed by Zwarenstein (1) and by Marlow and Koch (2). A study of the results which have been reported leads to the impression that the gonads (i.e. the gonadal hormones) have very little, if any, influence on the level of the serum calcium in mammals. In a recent communication, Riddle and Dotti (3), on the basis of results obtained by administering various estrogens to pigeons, doves, rats, and dogs, conclude that these substances, particularly theelin and dihydrotheelin benzoate, are potent agents in raising the serum calcium level. The actual increases in calcium level found by these investigators were small, except in the experiments on pigeons and doves. Furthermore, the data reported were obtained from very small groups of animals. On the basis of these results, Riddle and Dotti have suggested several implications relating the estrogenic substances, via the calcium balance, to sexual and racial differences in stature and to the "benefit currently derived from theelin administration in some mental cases." Also, the existence of a parathyrotropic hormone of the pituitary is questioned. The work of Riddle and Dotti was the first3 to indicate that the pure estrogenic substances cause an increase in serum calcium, and since such farreaching and important implications were made on the basis of this work it seemed desirable that a confirmation of the findings be attempted. We have consequently studied the blood calcium in rats, rabbits and monkeys before and after the administration of estrogenic material. The findings are presented in this communication. METHODS
The animals employed were placed on a constant diet, other conditions being maintained as constant as possible. The estrogenic material was injected subcutaneously as a sesame oil solution of either theelin or dihydrotheelin,4 specified in appropriate places. These two compounds, according to Riddle and 1 Aided by grants from the Committee for Research in Problems of Sex, the Nationl Research Council and 1from the Rockefeller Foundation, New York City. E. R. Squibb and Sons Fellow. 1 Saviano (4) is cited by Riddle and Dotti as having found increased serum calcium in 2 dogs as a result4 of treatment with progyno'n. The theelin and the estradiol benzoate (progynon-B) were furnished by the Schering Corporation, through the courtesy of Dr. Erwin Schwenk.
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Dotti, are the most potent of the estrogenic substances in ability to raise the blood calcium level. Control calcium values were obtained either before treatment with estrogen was begun, or from the blood of untreated animals taken at the same time as the experimental bloods. In one series of rabbits, and in the monkey series, both types of control values were obtained. Blood was always obtained by cardiac puncture without anesthesia, except in the case of rats which were lightly anesthetized with ether when the blood was taken. TABLE 1. EFFECT OF THEELIN ON RAT SERUM CALCIUM
Serum Calcium
Age at Litter Number
Body Wt. at Spaying gm.
I IA II VII IX X
143-156 125-149 171-206 175-191 184-191 178-208
Normal
Spayed
Normal
Spayed
days
days
mg. %
82 82 170 161 189 159
mg. % 12.32
mg. %
69
mg. % 11.92
12.5 11.3 12.6 12.1 12.9
12.5 12.1 12.2 11.9
13.0 12.2 13.3 13.0 11.9
12.3±0.3
12.2 + 0.1
12.7±0.3
157 141 169 139
Mean + mean dev. of mean
Non-Injected
2
Non-Injected III IV V VI XII
Injected1 6 days
First Injection
Spaying
2
12.0 12.8 13.7 12.3 12.5±0.3 1
Injected 12 days
11.7 12.8 12.8 12.5 11.6
12.5 12.7 11.8 12.4 14.1
12.8 12.8 13.4 11.9 12.8
12.2 14.3 14.0 12.4 13.5
Mean + mean dev. of mean
12.3 + 0.3
12.7±0.4
12.7±0.2
13.3±0.4
Grand Average
12.3
12.5
12.7
12.9
174-208 188-196 170-199 190-230 180-210
161 147 147 167 166
181 167 169 189 186
1 3
Injected animals received 100 y daily. Values obtained from pooled blood of 2 rats, the animals being too small to furnish individually sufficient serum for analysis.
Serum calcium was determined by the Clark and Collip (5) modification of the Kramer and Tisdale method. Duplicate determinations were made on each serum. The reagents were checked each time they were used. RESULTS
Forty-four female rats, 11 litters of 4 rats each, were placed on a mixed diet and kept under standard conditions for several days, and then 2 rats of each litter were bilaterally ovariectomized. Treatment with estrone in oil was begun 13 to 22 days later, one castrate and one intact rat of each litter receiving 100 Y of theelin daily. On the seventh day, after each of the injected rats had received a total of 600 f of theelin, blood was taken from half the litters, and the serum calcium of each rat determined. The remaining litters were continued as previously until each of the injected rats had received 12 daily injections of 100 y of theelin. On the 13th
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day, blood was taken from these animals and their serum calciums determined. The results of these determinations are given in table 1. As is obvious from an inspection of these data, the mean serum calciums of the 8 groups of rats are nearly identical. In only one case is an appreciable difference to be noted, namely, between the unoperated controls and the ovariectomized 12-day injected animals. Even this difference, 1.0 mg. per cent, would not usually be considered significant. Statistically, the value of 7'5 for this difference is 2.012, indicating a probability of approximately 8 in 100 that the difference is due to random sampling. It is therefore apparent that in TABLE 2. EFFECT OF ESTRADIOL BENZOATE ON SERUM CALCIUM OF OVARIECTOMIZED RABBITS
Control (Non-Injected)
3/8/37
Sample 2 3/15/37
15 days 4/13/37
31 days 4/29/37
mg. % 13.95 12.40 13.40 14.45 14.50 13.45 13.95 13.15 14.45
mg. % 14.55 14.75 14.00 13.55 14.75 13.65 14.80 12.80 13.65
mg. % 13.50 14.75 14.90 15.20 14.70 14.75 15.05 14.65 15.75
mg. % 14.45 15.30 13.70 14.55 14.90 14.40 14.90 14.40
14.06±0.23
14.81+0. 20
Sample 1
Rabbit 1 2 4
5 6 7 8
9 10
l
Mean + eM
13.74±0
24
13.90 + 0.17 1
Injected 1000 i.u. daily
14.55 14.57±0.15
14.69±0.12
e M = Mean deviation of the mean.
these experiments the serum calcium level was not influenced by the injection of theelin in relatively large doses. Since a great deal of work has been done on the effect of the ovary on the serum calcium of rabbits, and since the results of such work are in considerable disagreement, we decided to determine the effect, if any, of a crystalline estrogenic compound, estradiol, on the serum calcium of this species. The first experiment was carried out on a group of 9 adult ovariectomized female rabbits. The ovariectomies had been performed several months before, and the animals had been kept in the same cages and under the same standard conditions for an even longer period. The control serum calcium level was determined twice on each animal, one week intervening between the two determinations. Thirteen days after the last control blood was taken, injections of estradiol benzoate were begun, each animal receiving 1000 i.u. daily. The injections were continued for 31 days, the blood calcium being determined after 15 injections and after the complete course of 31 injections. The data obtained are given in table 2. 8 The probability that random sampling is responsible for a difference between 2 means is usually estimated from the ratio of the difference to its probable error. For small numbers of observations a more accurate estimate of the probability (P) is obtained by calculation of the value designated as '/' by Fisher ( 6 ) . A probability of 0.01 indicates one chance in 100 that random sampling is responsible for the difference between the two means.
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If one scrutinizes the data, it may be noted that the changes in the calcium values for the individual animals showed neither qualitative nor quantitative constancy. Some rabbits (No. 5,9, 10) show what appear to be increases in serum calcium after 15 days of treatment. On the other hand, rabbit 1 shows a fall in calcium of similar degree. Still others (No. 2, 6, 8) show practically no change. Similarly, when injections were continued for 16 more days, the calcium levels of some animals showed further increase, some a decrease, and others remained constant. It should be emphasized, however, that in all cases where apparent change occurred, it was relatively small. If the mean values are compared, it may be seen that the greatest difference (between the mean of the first set of control determinations and that of the 15-day injected group) is 1.07 mg. per cent, a difference which, in the light of the individual variations shown, would ordinarily not be considered of great significance. If these means be compared statistically, it is found that the differences between the first mean control value and the mean values after 15 and 31 days of treatment are possibly significant (P==
