THE FUNCTION OF THE PARATHYROIDS.”

BY HARALD A. SALVESEN.

(From the Physiological Institute of the University of Christiania, Christiania, Norway.)

(Received for publication, May 4, 1923.)

Though the parathyroids have been known for more than 40 years, their function is still unknown. In 1909 MacCallum and Voegtlin (1) formed the theory that the parathyroids control the calcium metabolism and by doing so exert an influence on the nervous system. This theory was based upon two important findings in parathyroidectomized dogs; namely (a) the benefi- cent action of calcium salts on the symptoms, and (5) the decrease in the calcium content of blood and tissues. They could also demonstrate an increased excretion of calcium in the first days following the operation and they maintained that all the symptoms were due to calcium deficiency. Voegtlin and MacCallum (2) later abandoned this view and returned to the previous theory that a poison is the cause of the symptoms following para- thyroidectomy.

The theory was abandoned by its authors chiefly because a rather concentrated solution of sodium chloride in large doses acted on the symptoms in the same way as calcium salt,s (3), contrary to what should be expected according to the experiments of Loeb (4), and because calcium salts, though checking the nervous symptoms for some time, failed to prolong the life of the parathyroidectomized animals.

The first obstacle against accepting the theory is probably removed by the recent work of Loeb (5) who showed that all

* These studies were aided by a grant from the Wright fund and the Freia Chocolate Factory’s fund for medical research of the University of Christiania. A preliminary report was published in the Proceedings of the Society for Experimental Biology and Medicine (Salvesen, H. A., Proc. Sot. Exp. Biol. and Med., 1922-23, xx, 294).

443

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444 Function of the Parathyroids

salts act in the same way in regard to permeability, but in very different concentrations. He found, for instance, that a M/S solu- tion of NaCl acts like a ~/1,000 solution of CaC12. What is true for permeability may also be true for irritability; in fact it was proved to be so by Joseph and Meltzer (3).

Concerning the objection that calcium salts fail to preserve the life of completely parathyroidectomized animals, it must be remembered that it is not known what is the fate of calcium injected into these animals; it may be eliminated again very rapidly. That calcium salts orally administered fail to preserve life may be due to bad absorption; it has never been shown that this calcium is absorbed at all. There are, moreover, many reports in the literature that calcium salts have preserved com- pletely parathyroidectomized dogs; the first report was made by Frouin in 1909 (6), a paper which seems to have been forgotten. The opponents of the calcium theory have always maintained that accessory glands have come into action and saved these dogs. The latest report is made by Luckhardt and Goldberg (7), who claim to have preserved the life of completely parathyroidec- tomized dogs by oral administration of calcium lactate.

In experiments which have been undertaken in this laboratory for 2+ years, we have succeeded in preserving completely para- thyroidectomized dogs by calcium administration for almost 2 years after the operation. It is the intention of this paper to show that calcium deficiency is the cause of the symptoms of parathyroidectomy and that the theory of MacCallum and Voegtlin (1) is probably right. This is shown by: (a) the chemi- cal findings of the blood in parathyroid insufficiency; (6) experi- ments to determine the fate of calcium salts injected in tetanic dogs; and (c) experiments on completely parathyroidectomized dogs preserved by calcium treatment. The chemical methods used in this work were the following: the’blood sugar was deter- mined by the method of Hagedorn and Jensen (8); the alkali reserve by the method of Van Slyke and Cullen (9) ; serum calcium by the method of Kramer and Howland (10); and in- organic phosphorus by the method of Tisdall (11).

1. The Blood Chemistry of Pamthyroid Insuficiency.-The ob- servation of MacCallum and Voegtlin (1) that blood calcium drops following parathyroidectomy was confirmed by Hastings

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Harald A. Salvesen 445

and Murray (12) who found that when the serum calcium was below 7 mg. per 100 cc. tetany occurred. Our own findings confirmed this. In seven partially parathyroidectomized dogs tetany never occurred, and blood calcium never dropped below 7 mg., but was usually about 8 mg. and in a few weeks was again restored to the normal value of 10 mg. All the completely parathyroidectomized dogs developed violent tetany (ten dogs), their serum calcium dropped and was, when tetany occurred, always below 7 mg. There seemed to be a certain relation between the degree of the lowering of calcium and the violence of the symptoms. The inorganic phosphorus of the blood serum increased considerably; after the initial rise the phosphorus might drop again, but still seemed ‘to be decidedly above the normal

TABLE I.

Dog lb. Complete Parathyroidectomy Aug. di, 193R.

Date. Weight. Blood Alkali Bugar. reserve

---

198 b. Pm cent p,:$,i

Aug. 19 17.00 0.087 63.4 “ 23 0.087 47.2

CS IllOF ganic P

--

mQ. mg.

10.00 3.0 3.50 12.5

“ 24 15.95 0.098 53.4 5.68 6.3

“ 26 15.65 0.104 63.7 6.54 6.5 “ 27 5.16 6.3

Rl3lUlhS.

Operation Aug. 21. Violent tetany. Calcium

chloride intravenously. No tetany. Calcium chloride

administration. No tetany. Mild tetany.

value. This confirms Greenwald’s (13) pioneer work of 1911 and 1913. The changes illustrated by Table I show the findings in one of the dogs.

2’. The Fate of Intravenously Injected Calcium Chloride in Para- thyroidectomized Dogs.-There are many reports in the literature of immediate relief of all the symptoms following injection of calcium salts in tetanic dogs. But the effect of the injection usually has been very short and the animals have died in spite of the treatment. In the present experiments, three of the completely parathyroidectomized dogs were treated with in- travenous injections of a 10 per cent solution of calcium chloride. A total of 2 gm. a day usually was injected. This checked the

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446 Function of the Parathyroids

symptoms, the dogs were completely normal for a while, but almost invariably there was violent tetany again tile next day which was checked by a new injection, and this was repeated day after day. It is easy to calculate that such doses of calcium chloride must raise the calcium content of the body fluids con- siderably. But whenever convulsions reappear after the injec- tion, the serum calcium always was found to be down to the same low level again, which shows that the injected calcium is disposed of in some manner.

To find what happens to the injected calcium, three of the tetanic dogs were given a fixed dose of calcium chloride intra- venously, and the urine and feces for 24 hours were analyzed for

J%?s. after inject& 0 2 4 6 8 10 12 14 16 18 20 22 24 9 0

&.c$ 8 %tidoci 2E,” - 6

-

FIG. 1. Calcium and phosphorus in serum following the injection of 1.6 gm. of calcium chloride intravenously in Dog 11, weight 14.5 kilos.

calcium by the methods of McCrudden (14). The previous day and the day of the experiment the dogs were given a calcium-free diet. The colon was rinsed out at the beginning and close of the period. The curves for the serum calcium and phosphorus were determined during this period.

Fig. 1 shows the effect of the injection of 1.6 gm. of CaC& on the serum calcium and phosphorus in one of the dogs. It is seen that 15 minutes after the injection the calcium was 15.6 mg. and decreased rapidly. 24 hours after, the calcium had the same

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Harald A. Salvesen

value as before the injection; that is, there had disappeared from the blood an amount of calcium exactly corresponding to the amount injected. There was also a rise in the phosphates. The curves for the other dogs closely resembled this curve. As Table II shows there was excreted an amount of calcium corre- sponding to the amount injected, but more than nine-tenths were excreted in the feces and less than one-tenth in the urine.

This rapid disappearance of the injected calcium may explain why the calcium treatment reported by previous workers failed to keep the animals alive. Only a few hours after the injection of a relatively large dose of calcium chloride the blood calcium is down to a level where tetany usually occurs. The experiments also show that in parathyroidectomized dogs there is a lowered threshold for the excretion of calcium through t-he intestines.

TABLE II.

Dog 11. Calcium Excretion in Urine and Feces. June 20 to 21,1922.

Urine Ca. Feces Ca. Total.

Qm. Qm. Qm.

0.042 0.504 0.546 Injected. . 0.578

3. Experiments on Completely Parathyroidectomixed Dogs Pre- served by Treatment.-Of ten dogs which were completely para- thyroidectomized (enough thyroid tissue was left to prevent cachexia), five died untreated within 33 days; one died 22 days after (the condition of this one was complicated by extensive necrosis of three legs following unsuccessful intravenous injection of calcium chloride); and two other dogs, also treated with cal- cium, were preserved. They were given 1 gm. of calcium chloride intravenously twice a day, but after a week or two the doses employed- could be lowered and at last omitted. During this time the dogs were fed a milk diet. Two other dogs recovered spontaneously during milk feeding by stomach tube; that this contributed to successful preservation will be seen from the fol- lowing experiments. These four dogs could be kept alive in- definitely on a milk diet; one lived for 21 months and was then killed; the others were either killed or, at will, brought into tetany from which they died. These dogs, which appeared

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448 Function of the Parathyroids

absolutely normal could, whenever it was wanted, be brought into tetany, usually within 24 hours, by changing the diet to meat. It is obvious that this condition formed an excellent opportunity for studying the various factors involved in the production of tetany.

TABLE III.

Dog 2. Complete Parathyroidectomy Mar. 14, 1921.

Date. Weight. s”u’,“B”:: &g. jJiJ: Remarks.

~__~-

1021 kg. per cent vol. prr cent VZ!l.

Apr. 6 10.73 0.099 52.5 5.22 Recovered after violent tetany, but still sick.

“ 26 10.75 0.098 54.3 5.87 Absolutely normal.

Though to all appearances normal, these dogs showed one characteristic finding in the blood: the cakiuva was still low. Table III shows an example of this. The calcium varied to some extent, but was usually between 5 and 6 mg. per 100 cc. of serum.

TABLE IV.

Dog 2. The Effect of Meat Diet en the Latent Tetanic Condition IS Months after Complete Parathyroidectomy.

Date.

1Lw2

Apr. 26

- ,

z

,

” 27 ‘I 28

11.00 a.m 7.30 p.m

kg. pm cent per ceni mu.

11.45 0.091 55.3 6.66 No symptoms. To all appear- ances normal. Meat diet started.

0.095 57.7 5.70 No symptoms.

11.45 0.099 47.6 5.28 Mild tetany. 4.96 Violent tetany. Injection of cal-

cium chloride. 11.45 5.56 No symptoms.

The inorganic phosphorus was usually at the upper border of the normal level or above. This low calcium is very significant and evidently characterizes latent tetany in contrast to cured

tetany.

Apr. 29

ieight, Blood Alkali

I I s”g&r. reserve Remarks.

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Harald A. Salvesen 449

(a) The Diet in Latent Tetany.-On a milk diet (minimum 500 cc. a day with bread or porridge) these dogs after the first critical period was over could be kept without symptoms for as long a time as desired. Meat always produced tetany (see Table IV), loss of appetite, and depression; prolonged meat feeding killed two of the dogs. It is an old experience that milk is beneficial and meat harmful to parathyroidectomized dogs. The reason for the difference will be seen from the following experiments.

(b) Why Milk Prevents Tetany.-Experiments with various forms of diet showed that it was the withdrawal of milk which produced tetany, not the meat diet in itself, and milk, therefore,

TABLE V.

Dog 1. The Effect of Milk, Poor in Calcium, 16 Months after Complete Parathqroidectomy.

In 100 cc. serum

Date. Weight.

19% kg.

Sept. 6 11.10 6.20 ii 7

“ 8

-

I -

Remarks.

Qm.

0.118 500 cc. calcium-poor milk + porridge. 0.039 No symptoms. Quiet, no appetite.

Food has to be given by stomach tube.

Fully developed tetany.

must contain a substance which prevents tetany. This sub- stance proved to be calcium, of which the milk used contained 1.2 gm. per liter, corresponding to about 10 gm. of calcium lactate. It was discovered, that when the amount of sodium oxalate calcu- lated to precipitate all this calcium was added to the milk, at least 90 per cent was carried down. Less than this was used in the experiments in order not to get an excess of oxalate in the milk, the precipitate was separated from the milk by centrifuga- tion, and this calcium-poor milk, which always was analyzed for calcium before feeding, was now useless in preventing tetany. Usually within 24 hours the dogs developed violent t,etany when fed this milk. Table V illustrates one of the experiments.

(c) Calcium Administration.-On the other hand, if an amount of soluble calcium salt was given corresponding to the Ca in the

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450 Function of the Parathyroids

amount of milk, which prevents tetany, the dogs remained normal on any diet; now when meat was given in excess, the dogs increased in weight and were normal in all their actions. The reason why meat produces tetany, therefore, must be due to the fact that it is very poor in calcium (see Table VI).

TABLE VI.

Dog 2. The E$ect of Meat Given with Calcium Lactate and Meat Alone, 163 Months after Complete Parathyroidectomy.

Date.

19ts

Aug. 26

Oct. 2 “ 3 9.45 a.m

6.00 p.m

In 100 cc. serum

ieight ~ CL%

k7. T7.

11.15 5.00

10.40 5.12

3.66

5.26

IniX- anic F

VW.

6.4

6.0

5.4

6.3

Remarks.

No symptoms. 350 gm. minced horse meat + 50 gm. fat + 4.6 gm. calcium lactate in 200 cc. water daily.

No calcium. Meat and fat as before.

Violent tetany. At 10 a.m. given 4.6 gm. calcium lactate in 200 cc. water.

Normal. Eats meat and fat with usual appetite.

TABLE VII.

Dog 18. The Effect of a Sin,gle Dose of Calcium Lactate upon Calcium and Inorganic Phosphorus of Blood Serum and the Clinical Symptoms.

In 100 cc. serum

Date. Weight C& InOT-

ganic P ____~

1982 ko . w7. w7.

Oct. 6 10.15 a.m. 15.50 4.39 6.9

2.30 p.m. 6.04 5.4

Remarks.

Violent tetany. At 10.45 a.m. given 9.2 gm. calcium lactate in 200 cc. water.

No symptoms.

It was possible by giving large amounts of calcium lactate by stomach tube to restore the calcium content of the blood almost to the normal level. From this condition it took a longer time than usual to produce tetany on a milk-free diet, and when tetany occurred the serum calcium was always found lowered again.

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Harald A. Salvesen 451

If tetany was produced from the latent stage a single dose of calcium lactate (5 to 10 gm.) usual‘ly checked all the symptoms. Analysis of serum calcium then always showed that at the moment

Grn.

x

150 - 4.3 B

0 reh Oct. Nov

MQ 10 17 22 act Oct. Nov 3 18 17

FOG. 3.

FIG. 2. Relation between serum calcium and glucose tolerance in Dog 2, weight 11.6 kilos. The upper columns represent the glucose tolerance; that is, the smallest amount of glucose given orally which produces glyco- suria. On Nov. 22, however, the glucose tolerance actually was lower than shown in the figure, as the urine after the administration of 50 gm. of sugar contained 10 per cent of glucose, and there were excreted 6.2 gm. of glucose in the urine.

FIQ. 3. Relation between serum calcium and glucose tolerance in Dog 12, weight 15.5 kilos. The tolerance on Oct. 3 actually was higher than indi- cated by the figure, as the amount of glucose given did not produce glyco- suria, and on Nov. 17 the tolerance was lower, as the urine after the adminis- tration of 80 gm. of sugar contained 8 per cent glucose, and there were excreted 4 gm. of glucose in the urine.

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452 Function of the Parathyroids

when the symptoms were relieved there was an increase in the calcium content of the blood, showing that it is the actual absorp- tion of calcium which cures the symptoms. Table VII illus- trates this. The same increase in serum calcium was seen when milk checked the symptoms.

Per cent 0.28

0.25

0.22

f; 0.19

Y t

; 0.16 0 0

;zi 0.13

0.10

0.07

1

1 2 3 4 5 Hours after glucose

administration

FIG. 4. Dog 2, weight 11.6 kilos. Blood sugar curve during glucose tolerance test. Oct. 17, 1922, 10.40 a.m., 120 gm. of glucose in 20 per cent solution were given by stomach tube. At 3.30 p.m. catheterized; urine contained 0.36 gm. of glucose.

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Harald A. Salvesen. 453

The fact that the blood calcium remained at a low level in these dogs might be interpreted as indicating that it is not the lowered calcium which is the cause of the symptoms; but the experiments in all four dogs showed that whenever the animals were brought into tetany from a symptom-free condition, the calcium was always st.ill further lowered; and whenever the ani- mals by administration of calcium or milk were made normal again, the blood calcium was always higher than during the symptoms of insufficiency. There seems to be an “adaptation”

TABLE VIII. The E$ect of Glucose Feeding on the Inorganic Phosphorus Content of Serum.

%:

12

2

-

--

Date. Veight

l.wd

Nov. 17 10.00 a.m.

12.40 p.m.

Nov. 22 11.00 a.m. 2.45 p.m.

kg.

13.5(

9.95

- I Per 100 CD.

SW”IXl.

- I

_

_

1

-

Ca

ml. mg. ‘er cenl

4.7 6.9 0.09s

4.1

5.4 4.9

0.204

2.52 0.111 0.227

IWX- mic P

Blood sugar. Remarks.

Tetany. At 10.30 a.m. given 80 gm. glucose in 20 per cent solution.

12.45 p.m. catheterized, urine contains 4 gm. glucose.

Tetany. At 11.15 a.m. given 50 gm. glucose in 20 per cent solution. 3.00 p.m. catheterized, urine contains 6.4 gm. glucose.

of some sort to the lowered calcium, but a further decrease promptly causes the characteristic symptoms. The inorganic phosphorus in these latent tetanic dogs showed no regularity except a certain tendency to keep at a high level, otherwise there was no relation between the phosphorus content of the blood and the clinical symptoms.

(d) Experiments on the Carbohydrate Metabolism.-Experiments made on two of these dogs showed that the tolerance for glucose (in 20 per cent solution by stomach tube) was distinctly lowered during the latent stage. When the dogs were brought into

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454 Function of the Parathyroids

tetany, the tolerance went down to very low values. On the other hand, when the blood calcium was increased by forced calcium feeding, the tolerance was markedly increased, and in one of the dogs it was impossible to induce glycosuria under these circumstances. When glycosuria developed the blood sugar curves went far above the threshold and they indicate that the lowered tolerance is not due to increased permeability of the kidneys but to a functional disturbance of the glycogen-forming organs. Figs. 2, 3, and 4 illustrate these experiments.

It was noticed that the inorganic phosphorus of serum might drop as a result of the glucose feeding (Table VIII). This obser- vation may support the theory of Embden and coworkers (15), that phosphoric acid plays an important role in the metabolism of sugar.

DISCUSSION.

From these experiments it is evident that the characteristic feature in the chemistry of parathyroid insufficiency is the drop in blood calcium which is the more marked the more parathyroid tissue there is removed. To this probably must be added the increase in the phosphates; and the quantitative experiments of Binger (16) indicate that there is a relation between the rise in phosphates and drop in calcium. But it is the drop in calcium which is the cause of the symptoms. This is shown by the fol- lowing facts: all the symptoms are relieved and the dogs made absolutely normal for a while by intravenous injections of calcium chloride; the symptoms reappear after a certain time, but not until the blood calcium again has reached the same low level as before. The injected calcium is very rapidly excreted through the intestines and only small amounts appear in the urine; even large doses disappear in a remarkably short time. This accounts for the fact that it has been so difficult to preserve parathyroidec- tomized animals by calcium injection.

It has further been shown that completely parathyroidectomized dogs can be preserved by calcium treatment, and this is a still further proof that the symptoms are due to the calcium decrease. In these dogs, which are in a state of latent tetany characterized by a low and very unstable blood calcium, and which require a milk diet rich in calcium to keep symptom-free, tetany can always

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Harald A. Salvesen

be produced by calcium withdrawal from the food. This is fol- lowed by a further drop in the blood calcium, and the dogs can always be saved by calcium administration. The relief of the symptoms is always accompanied by an increase in blood calcium. The hitherto unexplained fact that milk is beneficial to para- thyroidectomized animals, and, as has been shown in the present experiments, can protect latent tetanic dogs against tetany, is found to be due to the calcium content of the milk which makes it equal to a 1 per cent solution of calcium lactate.

The glucose tolerance experiments show that the supposed in- fluence of the parathyroids on the sugar metabolism is only an indirect one; the sugar tolerance sinks because the blood and body fluids are poor in calcium. When the blood calcium is restored almost to the normal level the tolerance for sugar is practically normal. That this symptom is due also to a lack of calcium further supports the calcium deficiency theory.

It is apparent from the experiments that in parathyroidec- tomized dogs there is a lowered threshold for the excretion of calcium in the intestines, and this is apparently the cause of the calcium deficiency and thereby of all the symptoms. It still remains to be explained how the parathyroid hormone prevents t,his unlimited excretion of calcium through the intestines. The be- havior of the blood calcium indicates that the actual recovery of completely parathyroidectomized dogs is not due to compensatory hypertrophy of accessory glands, as the action of these would be to restore the blood calcium to the normal level. The “adapta- tion” to a low calcium level, which in the beginning causes tetany, is not explained.

It will follow from these results that if a disease is due to para- thyroid insufficiency it will show a low blood calcium.

How lactose acts in preventing tetany as shown by Dragstedt (17) remains to be learned; determinations of blood calcium in these dogs preserved by Dragstedt’s method may give some information.

CONCLUSION.

The symptoms of parathyroid insufficiency are due to calcium deficiency. The parathyroids control the calcium level of the blood and by doing so they influence the function not only of the muscle and nerve tissues, but probably of all the organs.

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456 Function of the Parathyroids

The successful outcome of the operations is due to the valuable help of Professor Dr. 5. Torup to whom the author wishes to express his thanks.

BIBLIOGRAPHY.

1. MacCallum, W. G., and Voegtlin, C., J. Ezp. Med., 1909, xi, 118. 2. Voegtlin, C., and MacCallum, W. G., J. Pharmacol. and Exp. Therap.,

1910-11, ii, 421. 3. Joseph, D. R., and Meltzer, S. J., J. Pharmacol. and Exp. Therap.,

1910-11, ii, 361. 4. Loeb, J., Am. J. Physiol., 1900, iii, 383. 5. Loeb, J., J. Gen. Physiol., 1922-23, v, 231. 6. Frouin, A., Compt. rend. Acad., 1909, cxlviii, 1622. 7. Luckhardt, A. B., and Goldberg, B., J. Am. Med. Assn., 1923, lxxx, 79. 8. Hagedorn, H. C., and Jensen, B. N., Ugesk. Lager, 1918, lxxx, 1217. 9. Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 1917, xxx, 289.

10. Kramer, B., and Howland, J., J. Biol. Chem., 1920, xliii, 35. 11. Tisdall, F. F., J. Biol. Chem., 1922,1,329. 12. Hastings, A. B., andMurray, H. A., Jr., J. Biol. Chem., 1921, xlvi, 233. 13. Greenwald, I., Am. J. Physiol., 1911, xxviii, 103; J. Biol. Chem., 1913,

xiv, 363. 14. McCrudden, F. H., J. Biol. Chem., 1909-10, vii, 83; 1911-12, x, 187. 15. Embden, G., and coworkers, 2. physiol. Chem., 1914-15, xciii, 124;

1921, cxiii, l-312. 16. Binger, C. A. L., J. Pharmacol. and Exp. Therap., 1917-18, x, 105. 17. Dragstedt, L. R., Am. J. Physiol., 1922-23, lxiii, 408.

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Harald A. SalvesenPARATHYROIDS

THE FUNCTION OF THE

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