Alfred T. Shohl

Rickets in Rats. VI.∗ Effect of Phosphate Added to the Diet of Non-Ricketic Rats.:

Phosphate added to the diet causes rapid healing of rats made ricketic on a high calcium-low phosphorus regimen which has not been irradiated and is poor in vitamin D. This is evidenced by histological sections of the bones, blood serum analyses, bone analyses and studies of the metabolism of calcium and phosphorus. 1 As a result of the alteration in diet not only is the rickets cured but a transient tetany is also produced. The blood serum phosphate becomes extremely high—the calcium low. If the animals survive the tetany the blood serum calcium and phosphorus return to values considered normal in 2 weeks. Similarly, the feeding of phosphate to normal dogs is known to produce tetany (Salvesen, Hastings and McIntosh 2 ). When the diet for rats was altered by the addition of phosphate it was not considered a high phosphorus diet. It was known from the studies of McCollum et al. 3 to produce normal rats. The question accordingly arises whether non-ricketic animals shifted from a diet of low phosphorus to one of moderate phosphorus content would develop tetany. If so, would this be of short duration like that observed in the cure of rickets? Or is there something in the condition called rickets which predisposes to an exaggerated effect of phosphate? Plan—Rats fed with diets of a high calcium low phosphorus content and a liberal supply of anti-ricketic agents were compared to those fed on the same diet without vitamins. After 3 weeks on this diet all were given enough phosphate to make the Ca/P ratio 1/1. Seven and 14 days later the blood serum was analyzed for calcium and inorganic phosphorus, histological preparations of the bones were made, and the percentage ash in the fat-free bones was determined.

Rickets in Rats. V. Comparison of Effects of Irradiated Ergosterol and Cod Liver Oil.

Hess, in collaboration with Windaus 1 and others, have achieved the cure of rickets by minute amounts of irradiated ergosterol. Most of the studies on the cure of rickets in experimental animals are based upon the X-ray pictures, histological preparations of the bones, and blood serum analyses for calcium and phosphorus. Quantitative data are, however, supplied only by metabolism studies 2 , 3 , 4 and the analyses of the ash of the dry fat-free bones. 5 , 6 , 7 By use of technique described elsewhere 8 , 9 we desired to compare by the various analytical procedures the cure of standardized ricketic rats by cod liver oil and ergosterol. Animals fed on Sherman Diet B, when 28 days old were placed on Steenbock and Blacks high calcium-low phosphorus diet 2965 plus 10% lard. By analysis the diet contained 1.07% calcium and .178% phosphorus. The ratio of Ca/P was 6.0. They were kept on this regime for 21 days. Then the diet was altered by replacing 2% of the lard by cod liver oil, or irradiated ergosterol in olive oil.† Both were fed separately and not mixed with the diet. The amount of ergosterol represented .01 mg. per rat per day. The animals were killed after 14 days on the curative diet. The autopsies showed intramuscular hemorrhages in the upper hind extremities of 2 of the rats which received ergosterol. The rats were studied as to (1) blood serum analyses, (2) histological examination of the bones, (3) bone analyses, and (4) metabolism of calcium and phosphorus.

A Rat Board for X-Ray, Photography, and Operative Procedure.

The board for holding rats, shown in the accompanying illustration, will be found convenient for x-ray exposures, photographs or for an operating board. It was designed to obviate the danger to the workers of exposure to x-rays. We calculated that the number of exposures necessary was in excess of the dosage compatible with safety. With the present device, one person can do the work for which 3 were formerly required. The animal is placed in the holder outside the x-ray room and while the exposure is made, a second animal is made ready. Thus no worker is exposed to x-rays. The apparatus shown was made from sheet aluminum. It can be made of steel or covered with lead as desired. The clips are battery clips with the teeth flattened to the plane of the clip and guarded with rubber tubing. The spring of the clip should not be strong enough to hurt the animals paw. A test on the finger will determine whether the jaws should be bent or the spring altered. The clips are fastened to the frame with coiled spring wire. This permits the animal to be held firmly but is still elastic. Under such conditions the animals are restrained but not immobilized. They do not feel frightened and remain quiet and docile upon the board. Mr. B. Dan, Supervisor of Equipment of the Institute of Pathology, Western Reserve University, has given suggestions for some mechanical features and his help is gratefully acknowledged.

Equilibria involving calcium, hydrogen, carbonate, bicarbonate, primary, secondary and tertiary phosphate ions

The complete equilibrium involving CO3 = and PO4 = takes place when the solution is made more alkaline. This relationship may be derived from the equation. The above equation for the complete equilibria is not applicable until better values for the third dissociation constant are available. Therefore, the values may be better derived from the simpler relationship for K7. This gives a value for the equilibrium in terms of Ca++, HCO3 -, HPO4=, and H+. This value calculated theoretically from known values of the intermediate constants was found to agree well with the experimentally determined value reported elsewhere.1 The concentration of the ions could be determined from analysis of all the salts in the system and the calculated degree of ionization. In systems in which this is not possible, due to the presence of unknown compounds as in blood serum, the CO2 from bicarbonates and H2CO3 the P from inorganic phosphates and the H+ must be determined. The only unknown is the Ca++ which may then be calculated from the equation.

The quantitative determination of the alkali retention in growth

In a report made last year 1 on acid base metabolism in infants, a method was proposed for measuring the retention or the loss of alkali in the body. The method consists in determining the equilibrium or balance of all the acid and basic radicals. The values are computed in terms of normal solutions and the excess of base retention or excretion can then be stated in terms which are a common denominator for all of the elements in mineral metabolism. It is necessary to determine the acids and bases in the food, urine and feces. The results calculated in terms of normal solutions are then totaled. By suitable methods of titrating the urine and stools, approximately the same values are obtained as by the method of analyzing the individual elements. The work will be reported in extenso in a forthcoming number of the American Journal of the diseases of Children, including methods and protocols. The metabolism experiments show that for an infant, A, weighing 9 kilos, and one, B, weighing 51/2 kilos, fed on cows milk, water and sugar, the positive base balance, as shown in the accompanying figure, was 98 c.c. 0.1N alkali in excess of acid and 64 c.c. 0.1N alkali in excess of acid. Calculated on the basis of alkali retained per kilogram of body weight, this equals 11 c.c. O.1N alkali retained per day in both cases. A recalculation of the available data in the literature confirms our results. This appears to be a very large amount of base retention per day. However, when the values are computed it is found that of this alkali retained, 2 c.c. is required for the protein increase, 4 c.c. for the alkali reserve and about 57 c.c., by far the greatest part, for the building of bone.