David H. Shelling

Effect of dietary calcium and phosphorus on toxicity of lead in the rat; rationale of phosphate therapy.

Since the introduction of calcium salts or calcium containing foods, by Aub and his associates, 1 in the treatment of lead poisoning, numerous papers have appeared in the literature advocating such therapy. The rationale of the procedure was based on the following facts: First, the solubility of lead phosphate was found to be analogous to that of calcium phosphate; hence Aub and his associates believed that lead might be deposited in the bones in the same manner that lime salts are and thus be removed from the circulation. Second, when diets low in calcium were fed to cats, the trabeculae of the bones of the animals were diminished in size and in number as compared to those of animals fed diets to which calcium had been added. This was interpreted to mean that the addition of calcium salts to diets in general results in an increased storage of lime salts in the trabeculae; and since lead was supposed to behave like calcium, its deposition in the bones could be hastened or increased by furthering the process of calcification through the administration of calcium. Third, the fact that lead colic may be alleviated, almost instantly, by the intravenous administration of calcium chloride was thought to add additional evidence that calcium “drives” lead into the bones. However, the later studies of Aub and coworkers 2 discredit such an assumption since the alleviation of pain is too rapid to be due to precipitation of lead in the osseous tissue. Aub and his .coworkers are, therefore, now inclined to believe that the action of calcium in this instance is to relax the intestinal musculature. That the administration of calcium salts does not always result in improved calcification unless the phosphorus in the diet is controlled, may be inferred from the experiments of McCollum, Shipley and Park 3 on the production of rickets in rats.

Relation of Calcium and Phosphorus of Diet to Toxicity of Viosterol.

The discrepancies apparent in the literature as to the toxicity of viosterol may be attributed to (1) differences in the potency of the viosterol, (2) the character of the diet in regard to its content of calcium and phosphorus, (3) the age of the animals at the beginning of the experiment and (4) the duration of the experiments. Thus, many published results are based on the use of viosterol of unknown potency and varying methods of assay; and the diets employed range between an undescribed so-called “normal diet”, through the Steenbock and McCollum rachitogenic diets, to diets consisting of “table-left-overs”. In the present study an attempt was made to ascertain the relation of calcium and phosphorus of the diet to the toxicity of viosterol. The rats used were of our own breeding stock and were put on the experimental diets at the time of weaning. The viosterol was incorporated in the diet; the dosage was calculated in cod liver oil equivalents, i. e., the amount of viosterol which produced a line test in 5 days, when added to the Steenbock rachitogenic diet, equaled 1/4% of the diet in terms of cod liver oil potency. (a) Steenbock-Bills stock diet. Ca = 0.515, P = 0.450 gm. %. The toxicity, duration of life and well-being of animals on this diet are directly proportional to the dose of viosterol. 40,000 to 80,000 times overdosage is very rapidly fatal, sometimes with little evidence of metastatic calcifications, especially the 80,000 times overdosage. On 10,000 and 20,000 times overdosage animals do well at first, then they begin to decline in health and finally die, showing calcification of most organs, especially of the vascular system. Those on 2,000 and 4,000 times overdosage were killed at 10 months.

A Simple Method for Production of Vitamin D Milk of Known and Controllable Potency

Since the introduction of irradiated ergosterol and irradiated foods for the prevention and treatment of rickets, several methods have been employed in order to impart anti-rachitic properties to milk. Among these may be mentioned: (1) the irradiation of milk in both the powdered and liquid forms; (2) irradiation of the cow; and, (3) feeding to the cow substances containing large amounts of vitamin D, as for example cod liver oil or irradiated yeast. The irradiation of the cow as a means of imparting vitamin D to the milk is obviously most impractical from the commercial point of view, as is also the feeding of large amounts of cod liver oil. The irradiation of the milk itself has the practical disadvantage of being expensive since it involves the installation, maintenance and operation of expensive apparatus in the farm or dairy where it is employed. The direct irradiation of the milk and the indirect method of feeding irradiated yeast to the cow both have the disadvantage that they are not absolutely reliable methods of holding the vitamin D content at fixed concentration, so that repeated testing of the milk at regular intervals becomes necessary in order to be sure that the vitamin D content of the milk has not fallen below the standard. In view of these difficulties it occurred to us that the method for adding fats to milk, which has been employed successfully by Holt, Tidwell and their associates, in our laboratories, for the past 4 years, might also be more practical for fortifying milk with vitamin D. Our procedure consists simply of homogenizing, in water, a concentrated oily solution of viosterol of known potency, with the aid of an emulsifying agent. We have tried several emulsifying substances, which are quite feasible as such, but in order to produce an emulsion which will be stable for months or years under ordinary environmental conditions, we have found lecithin to meet this requirement. This stable emulsion appears white as milk, is miscible with it, and has no objectionable odor or taste.

Effect of Viosterol on Serum Calcium of Parathyroidectomized Rats.

Recently Hess 1 suggested that the effect of viosterol in raising the serum calcium level was through the parathyroids. This suggestion was based on his observations that in monkeys and dogs fed large doses of viosterol the serum calcium level frequently rose to 13-16 mg. %. After thyroparathyroidectomy, however, large doses of viosterol failed to raise the calcium above the tetanic level. Greenwald 2 reports similar observations with cod liver oil and irradiated ergosterol. On the other hand, Jones, 3 Brougher, 4 Urechia and Popovicius 5 report that antirachitic agents, when given in large doses, are able to raise the serum calcium level and ameliorate the tetanic symptoms in thyroparathyroidectomized animals. The diets in these experiments are either not mentioned or are of an inconstant composition. Greenwalds diets were usually of the high phosphorus and low calcium type, and he attributes the beneficial results of vitamin D in one of his animals to an accidental mixture of calcium in the infusorial earth and that in Broughers experiments to the administration of milk. In the preceding paper 6 the importance of the calcium and phosphorus of the diet in relation to the serum calcium level after parathyroidectomy was stressed. In the present communication the effect of viosterol and of diet on parathyroid tetany is reported. The results may be summarized as follows: 1. Low calcium and optimal∗ phosphorus diet. (a) When viosterol is added to this diet in dosages of 5% equivalent to cod liver oil† moderate elevation of the serum calcium may be observed after 20 days, and the animals continued on this diet with mild or no tetany for several months. The serum calcium levels ranged between 6.9 to 8.1 mg.%.

Effect of Diet and Viosterol on Calcium Deposition in the Callus of Parathyroidectomized Rats.

Canal 1 showed that calcium fails to be deposited in the callus following fracture in parathyroidectomized rats, and Morel 2 noticed a similar defect in cats. Erdheim 3 concluded that the deficient calcification in the callus throughout the skeleton of parathyroidectomized rats resembles that of rickets and osteomalacia. The above experiments were conducted when exact knowledge of dietary requirements were wanting and hence, there is a possibility that the diet may have contributed to the failure of calcification in the callus in the presence of a parathyroid deficiency. The following experiments were conducted to ascertain the effect of alterations in the calcium and phosphorus diet and of viosterol on callus calcification. The procedure and diets have been briefly described. 4 The radii and ulnae of rats were fractured while under amytal anaesthesia. The rats were placed on a low calcium diet until tetany developed and then they were divided into groups according to the diet they were to receive. X-rays of the fractured bones were taken weekly until the rats were killed. The results may be summarized as follows: 1. (a) Low calcium diet. In spite of the low calcium diet the calcification of the callus progressed moderately within 30 days as evidenced by X-ray. The serum calcium in this group remained low and the animals continued to have tetany. (b) When 1% CaCO3 was added to the diet, the calcification in the callus seemed somewhat denser. (c) Stock diet. Very good calcification of the callus was observed within 20 days. (d) Steenbock rachitogenic diet. In spite of its being a ricketsproducing diet, the calcium deposition in the callus was marked. (e) High phosphorus and low calcium diet. The deposition of calcium salts was very poor, even after a month.

Effect of adrenalin upon blood sugar following ligation of the hepatic artery.

In a previous communication, 1 we demonstrated that exclusion of the arterial supply to the liver by ligation of the hepatic artery and its collateral branches causes death in hypoglycemic convulsions within 15 to 60 hours, depending upon the amount of glycogen previously stored. The present experiments deal with the effect of adrenalin upon the blood sugar level following this procedure. Five-tenths to 1.5 cc. of 1/1000 solution of adrenalin was injected intravenously at varying periods following ligation of the hepatic artery in dogs. The following observations were made: 1. The degree of hyperglycemia following adrenalin injection varies inversely with the period of time following ligation of the hepatic artery; i. e., the longer the time permitted to elapse after ligation the less the increase in blood sugar. 2. When the animal develops all the manifestations of hypoglycemia, adrenalin no longer influences the blood sugar level. 3. When adrenalin fails to cause an increase in blood sugar, hypoglycemic convulsions and death may be predicted in 2 to 5 hours. 4. When adrenalin had no effect on blood sugar level, the tissues of these animals, examined at death, showed complete absence of glycogen. This is in agreement with the results of the work of Ringer, 2 who showed that in a phlorhizinized diabetic dog, totally depleted of glycogen by shivering, adrenalin does not alter the D :N ratio, through an elimination of extra sugar. 5. The blood sugar level does not necessarily indicate the quantity of glycogen stored in the organism. In Dog 20, 1 cc. of adrenalin, intravenously, had no effect upon the blood sugar, although the blood contained 82 mg. sugar per 100 cc. One hour later this animal was in hypoglycemic shock with a Mood sugar of 52 mg. Dog No. 15 showed no effect of adrenalin upon the blood sugar, although the blood sugar was 90 mg., and in dog No. 57,4 1/2 hours before convulsions and death of the animal, the adrenalin had no effect on the blood sugar level at 86 mg.

Role of the Parathyroids in Calcification, and Susceptibility of Parathyroidectomized Rats to Viosterol.

The long continued administration of parathyroid extract to animals results in an increased excretion of calcium and phosphorus from the body. 1 , 2 , 3 Similarly, the presence of hyperfunctioning parathyroid glands results in a negative calcium and phosphorus balance and bone decalcification termed clinically, osteitis fibrosa cystica. Several observers noticed marked improvement when one or more of these glands was removed. Hence, in hypofunction a marked retention of calcium and phosphorus should result. Greenwald, 4 contrary to previous assumptions, demonstrated retention of these elements after thyroparathyroidectomy in dogs. The validity of Greenwalds conclusions may be questioned because the thyroid extirpation may have contributed to this retention. However, in metabolism experiments on rats, in which the parathyroids alone were removed, retention of calcium and phosphorus was observed, provided the diet contained a certain amount of these elements, 5 thus substantiating, in part, Greenwalds hypothesis. However, no attempts have as yet been made to determine the depots of retention of calcium and phosphorus. That their deposition is not always in the osseus tissue is evidenced by the observations of Erdheim, 6 Iselin, 7 and Toyofuku. 8 Leopold and Von Reuss 9 found that the combined soft tissues of parathyroidectomized rats contained more calcium than those of control rats. In the course of study on the effect of calcium, phosphorus and viosterol intake on parathyroidectomized rats it was noted that they were much more susceptible to viosterol hypercalcification than normal animals. A dose of viosterol, which ordinarily would not produce calcification in a normal rat in a given length of time, may cause calcification in the organs of the parathyroidectomized animals, provided certain amounts of calcium and phosphorus be present in the diet.

Fractionation of Irradiated Cholesterol Antirachitic Potency of the Fractions.

There have been a number of attempts to separate an antirachitically potent fraction from cholesterol irradiated in air. Hess, Weinstock and Sherman, 1 and Rosenheim and Webster 2 tried without success. The latter authors claim to have isolated an active fraction from cholesterol irradiated in nitrogen, but their experimental data do not seem to warrant such a claim. Nitzescu and Popoviciu 3 reported the separation of irradiated cholesterol (presumably irradiated in air) into an active and an inactive fraction. The preceding paper 4 is a report of the separation of a yellow oil formed from cholesterol as a result of irradiation in air. The antirachitic potency of this “U. V. oil of cholesterol” was tested by means of curative experiments on rachitic rats, in which the material tested was incorporated in the diet. At the May, 1926, meeting of the American Pediatric Society, one of us, in discussing a paper by Schlutz and Ziegler, reported 5 that this material cures rickets in rats. During this same discussion, Julius H. Hess 6 stated that Koch had obtained an active extract from irradiated cholesterol. The present communication is a brief report of the results so far obtained. The new points brought out by this investigation, in which more than 100 rats were used, are as follows: 1. Crude “U. V. oil of cholesterol” cures experimentally produced rickets in rats. 2. Cholesterol-free “U. V. oil of cholesterol” cures experimentally produced rickets in rats. 3. The antirachitic effect of the U. V. oil is not due to material extracted by ether from the reagent employed for the removal of the cholesterol.

Antirachitic Properties of Cod Liver Oil Concentrate.

Cod liver oil causes retention of lime salts and also cures and prevents rickets when administered orally. The assumption, therefore, was made that the beneficial effects of this agent result from its acting directly on the mucosa of the gastro-intestinal tract. Thus, Zucker and Matzner 1 were unable to obtain healing in rachitic rats with a subcutaneous injection of an extract of cod liver oil, although when administered orally it was invariably-potent. Similarly, Lesne and Vogliano 2 failed to cure rickets in rats when cod liver oil was injected hypodermically. On the other hand, Soames 3 reported moderate healing of rickets when cod liver oil was injected either subcutaneously or intraperitoneally. Hess, Weinstock and Hellman 4 were successful in curing rickets with an aqueous suspension of irradiated cholesterol administered subcutaneously; and Wilkins and Kramer 5 have shown that an ether solution of a concentrate of cod liver oil is capable of curing severe rickets in children. The experiments briefly reported here may shed some light on the discrepancies in the literature just mentioned. The results of these experiments, in which 60 rats were used, are as follows: 1. The unsaponifiable fraction∗ of cod liver oil from which most of the cholesterol had been removed (crude concentrate) cured experimmtal rickets within 14 days when injected subcutaneously in ether solution. The same result was obtained when it was incorporated in cottonseed oil and added to a rickets-producing diet. The curative dose was equivalent to 2 cc. of cod liver oil. 2. When entirely freed from cholesterol by means of digitonin precipitation, an active concentrate was obtained which cured experimental rickets both when fed, and when injected in ether solution. The curative dose was equivalent to 2 cc. of cod liver oil.

The effect of ligation of the hepatic artery on carbohydrate metabolism.

Conclusions (1) Ligation of hepatic artery of dogs causes death in hypoglycemic convulsions. (2) The period of survival after ligation depends upon the amount of glycogen previously stored, and varies between 15 to 60 hours. (3) There is a total depletion of glycogen in the tissues at death. (4) Administration of glucose prevents convulsions and prolongs life for several hours. (5) Severing the plexus of nerves surrounding the hepatic artery does not produce this hypoglycemic syndrome.