The following are established as specific actions of vitamin D in the body:
Action No. 1
Vitamin D is required for normal growth in mammals. This is probably related to calcium and phosphorus absorption and utilization. When the rate of bone growth is below normal, as is the case in vitamin D deficiency, the rate of body growth is likewise retarded: A deficiency of dietary calcium or phosphorus will also result in subnormal growth. It appears that the effect of vitamin D on growth is closely related to its effect on bone development.
Action No. 2
Vitamin D increases calcium and phosphorus absorption from the intestine. In vitamin D deficiency the fecal calcium and phosphorus excretion are reduced after the administration of the vitamin. The urinary excretion may be increased also, but usually to a lesser extent. The end result in such cases is a "net" gain in these elements or a retention by the body. A negative balance of these elements is brought into equilibrium or into a positive balance.
The mechanism here remains obscure. The net gain is not due to a decreased reexcretion of calcium into the gut, but to an absolute gain in absorption. A change in the pH of the lower intestinal tract as a result of vitamin D has been suggested as one factor. Greater acidity increases the solubility of calcium salts, such as the phosphates, and this should lead to increased absorption. Studies with isotopic Ca (Ca45) have not added pro-foundly to our knowledge of absorption. Harrison and others showed increased calcium transfer through the intestinal wall in experiments using Ca45 with everted intestinal sacs.
Schachter arid co-workers found that in rats the calcium transfer is greater in proximal than in distal segments of the small intestine and also that calcium transfer is more readily performed by intestinal tissue of young growing rats than that of older animals. It appears that vitamin D increases the efficiency of Ca absorption only under conditions in which the intestinal Ca is poorly soluble.
Increased absorption as soon as one hour after Ca45 by stomach tube in the rat has been observed. Maximal absorption ofCa45 resulted from 10 IV of vitamin D in the rat, although a hundredfold increase in vitamin intake resulted in further elevation 'of blood Ca45• This increase was apparently due to the action of vitamin D on bone salts. Bile salts are concerned in some way with calcium absorption, and vitamin D is thought by some workers to increase the activity of these molecules in enhancing calcium absorption. Certainly other .mechanisms, more subtle than these, must be in operation.
Action No. 3
Vitamin D is antirachitic, Rickets is a disease of the young. The disease may involve a low blood calcium level or a low blood phosphorus level. In humans the latter type is generally seen. Either can be produced in animals by dietary means. In infancy the inorganic phosphorus level (primarily as HPO 4- and H2PO 4- ) of blood is normally 4 to 6 mg per cent. In rickets this may be decreasec to 1 or 2 mg per cent.
At the ends of bones during normal growth the osteoblasts, or bone-forming cells, appear as the cartilage cells degenerate and disappear. After capillaries grow into this site, the osteoblasts deposit bony matrix. The process is a continuous one in that new osteoblasts are always under formation. In a vitamin D deficiency the cartilage cells do not degenerate but continue to grow; consequently, capillaries and osteoblasts are not formed.
The cartilage tissue increases in size and remains uncalcified. In more severe defiCiency bone mineral may be resorbed, leaving a greater area of osteoid tissue. The time and the degree of the deficiency obviously determine the extent of the abnormality. In infants and children clinical symptoms of severe deficiency are readily seen on gross examination.
Enlargements of the ankle, knee, and wrist joints are noted. Other prominent features are bowed legs, delayed closure of the fontanelle, beading of the ribs at the costochondrarjunction
rachitic rosary"), and delayed tooth eruption. The mineral content of bone decreases as the severity of the rickets progresses. The ash on a dry, fat-free basis may reach one half to one third or less of the normal value. The administration of vitamin D to rachitic animals brings about the degeneration of cartilage cells, the growth of capillaries in this area, and ossification.
How does the presence of vitamin D bring about such remarkable and rapid effects? Here again our information on actual mechanisms is almost nil. The increased absorption of calcium and phosphorus from the gut due to vitamin D is not the answer, since healing of rachitic bones in animals can take place without food or with food devoid of these minerals. Also, the intravenous administration of calcium and phosphorus salts to raise blood levels is not effective in initiating healing. The administration of parathyroid hormone or of AT 10, both of which increase blood calcium, likewise produces little or no antirachitic effect. It was demonstrated in 1928 by Hess that when rachitic bone is placed in blood serum from rachitic animals no change takes place, but if serum from a normal animal is used, calcification is initiated. Robison demonstrated the presence of an enzyme capable of splitting inorganic phosphate from organic combination (phosphatase).
Various phosphatase enzymes are now known to be in the body. In bone cartilage and especially in rachitic osteoid tissue the concentration is high. This incidentally increases the blood content of the enzyme. The level is of value in diagnosing early rickets and some other bone abnormalities. Many investigators have found rather good agreement between vitamin D deficiency and increased blood alkaline phosphatase. In a study of a large number of children, aged six months to 2.5 years, a mean value for the serum phosphatase of 9.4 Bodansky units was reported for the normals, whereas the rachitic children had in general over twice this phosphatase activity. It was also indicated that the increased enzyme activity correlated well with severity of the clinical symptoms of rickets.
Apparently the enzyme is responsible, under normal conditions, for liberating inorganic phosphate from organic combination and thus increasing at the site of ossification the ion product Ca++ x P04---. It has been suggested that bone is deposited in the matrix only when the solubility product of Ca+ + X P04--- in plasma is exceeded, and perhaps that the calcium and phosphate ions must be present in a special form. At any rate the process of ossification requires phosphatase enzymes, and it now appears that a number of other enzymes are likewise intimately involved in the process.
The glycolytic process with the many enzymes involved not only may provide some as yet unidentified organic phosphate ester as the substrate for Rhosphatase, but may also supply the energy requirements. Rachitic bone contains glycogen and the enzymes required for the glycogenolysis process. When such bone slices are placed in the proper medium containing inorganic phosphate, calcification takes place. However, if phlorizin is also added, calcification does not proceed. This substance is known to inhibit phosphorylase, the enzyme responsible for the conversion of glycogen plus inorganic phosphate into glucose-1-phosphate.
The inference is that this block precludes the further steps leading to the required phosphate ester which might act as phosphatase enzyme substrate. Upon the addition of
glucose-I-phosphate, the compound thatthe system is unable to produce, calcification proceeds in the presence of the inhibitor. By studying inhibitors of other enzymes in the system, the conclusion has been reached that phosphorylative glycolysis plays an important role in the calcification process.
It is likely that normal bone calcification in vivo likewise involves such complicated mechanisms to supply a special type of phosphate ester in order that phosphatase may liberate phosphate ions at the active calcifying site. For'.the normal bone formation or for calcification of rachitic bone, vitamin D is required. What part it plays is still questionable. Its activity may involve some effect on the phosphatase enzyme system, or the state of serum calcium or phosphorus or both, or the various cells concerned with laying down bone.
Phosphorylated vitamin D, but not the unaltered molecule, was shown to have a marked initial activating effect on kidney alkaline phosphatase. What part, if any, this might play in bone formation is not now apparent. The rat differs from other species in vitamin D requirement. A rachitic condition is easily induced in this animal by keeping him on a diet with an upset calcium-phosphorus balance, and bone healing will proceed in the absence of Vitamin D if the animal fails to grow or loses weight. This will be discussed further under the section on vitamin D assay. Without understanding each biochemical event in calcification, the process may be visualized as follows: As cartilage cells in the growing end of bone degenerate, the matrix is invaded by capillaries and the bone-forming cells called osteoblasts. These cells may induce the seeding of crystals of some form of calcium phosphate.
The collagen fibers of the matrix are regularly bonded with a spacing of 640 A, and this spacing appears ~ be critical and probably involved also in the seeding process. These workers regard alkaline phosphatase as essential in splitting organic phosphate esters to increase the Ca tims P product to a critical level. The initial precipitate is unstable and changes to hydroxyapatite. Maturation consists in crystal growth and displacement of water among other changes. A more dense bone is thus produced.
Action No. 4
Harrison and Harrison demonstrated a specific function of vitamin D on kidney tubular reabsorption of phosphate, They developed rickets in dogs and then by phosphate clearance studies showed that the administration of large doses of vitamin D definitely increased phosphate reabsorption. At equilibrium the plasma phosphate .was thus elevated, and these workers postulated that such an effect is probably part of the antirachitic action of vitamin D. Such an action is definitely antirachitic since it tends to conserve phosphate and to increase the Ca++ x PO 4-- - product of the plasma. It has been pointed out that the increased tubular reabsorption may be a secondary effect to the primary increase in circulating calcium.
Action No. 5
Citric acid is a normal constituent of many body tissues, including bone. Rachitic rats given vitamin D show increased urinary excretion of citric acid and increased levels in blood, bone, kidney, heart, and small intestine, with no elevation in liver. Such findings indicate a rather general effect of the vitamin on citric acid metabolism. Steenbock and co-workers have continued with in vitro enzyme studies and demonstrated that addition of vitamin D to either a rachitogenic or a nonrachitogenic diet resulted in a depression of in vitro citric acid oxidation by kidney homogenates or mitochondria. In further experiments the addition of the vitamin to an in vitro system of kidney mitochondria reduced citrate oxidation. These findings help explain the increased tissue citrate levels in a deficiency of the vitamin.
Action No. 6
It has been suggested that vitamin D increased the activity of the enzyme phytase in the rat intestine. This enzyme hydrolyzed food phytic acid (grains primarily), yielding inorganic phosphate. More phytic acid is excreted in the feces of rats and dogs in a deficiency state than when the vitamin is given. The rat intestine produces phytase, and in rachitic rats phytic acid of a high-cereal diet is completely hydrolyzed only when vitamin D is given. However, the increased enzyme activity does not liberate sufficient inorganic phosphate to account for the antirachitic action of the vitamin. Vitamin D is known to stimulate the release of calcium and strontium from isolated rat kidney mitochondria.
The vitamin D effect on release of calcium is observed only in the presence of an oxidizable substrate such as succinate and is accompanied by a release of inorganic phosphate. Thompson and DeLuca showed that rats fed a diet with added vitamin D showed a threefold increase in the incorporation of P32-orthophosphate into the phospholipids of intestinal mucosa. At the same time there was no alteration of the incorporation into nonlipid organic phosphates. Such findings do not readily fit into a biochemical scheme at the present time. It is advisable at this point to digress and bring the parathyroid glands into the discussion. One of the primary actions of the parathyroid hormone is to increase the urinary excretion of phosphate and concomitantly reduce the plasma phosphate level.
It is known too that an increased plasma calcium level tends to lessen the activity of the parathyroid glands and that a low level results in increased activity. On the basis of these two facts and on an imposing accumulation of data of their own on the action of vitamin D, the parathyroids, etc., on calcium and phosphorus metabolism, Albright and Reifenstein interpreted the results of Harrison and Harrison on the basis that the increased kidney reabsorption of phusphate resulted from decreased parathyroid activity (from increased blood calcium level). Albright and co-workers showed that in a patient with idiopathic hypoparathyroidism the administration oflarge doses of vitamin D increased urinary calcium and phosphorus excretion more than the decrease in fecal calcium and phosphorus excretion. The extra mineral obviously came from bone, and negative balances of these two elements developed.
Albright feels that a primary action of vitamin D is to increase the urinary excretion of phosphate, but that the action is quantitatively far less than that of the parathyroid hormone. In the case of an individual with a normal parathyroid, he feels that this action is masked by the ability of the vitamin to increase the absorption of calcium from the intestine, leading to an increased serum calcium level, which inhibits parathyroid hormone production, resulting in lessened urinary phosphate excretion. Under these conditions the serum phosphate increases, whereas in the individual with a parathyroid hormone deficienCy the end result on plasma phosphate is opposite after the administration of vitamin D. Increased blood calcium cannot have an effect on a nonfunctional parathyroid gland. The other primary action of the parathyroid hormone (besides increasing urinary phosphate exc-retion) is to increase a low blood calcium level or maintain a normal one by removal of calcium from bone.
Excess hormone causes hypercalcemia. As a result of the two established actions of the hormone, two schools of thought on its primary action have developed. One group holds that parathyroid hormone brings about increased phosphate excretion by the kidney and that changes in blood calcium and phosphate levels are secondary to this. A variety of experiments indicate increased phosphate excretion, followed by elevation of the blood calcium, upon administration of the hormone. The other school claims that parathyroid hormone brings about dissolution of bone calcium and that the renal effects are secondary.
Evidence in favour of this theory is found in the various experiments in which nephrectomized animals, unable to excrete phosphate, respond to the hormone with an increase in blood calcium. In one such experiment, nephrectomy (rats) appeared to nullify the effects of the hormone upon blood phosphate levels while the 'action as regards blood calcium was maintained. These workers were led to postulate that normally the hormone has a direct and independent effect on both calcium and phosphorus metabolism.
In a review on the mode of action of the parathyroid hormone the action of maintaining blood calcium level is stressed, and new fmdings do not offer an explanation of how this is accomplished. It is advisable at present to keep in mind the two well-established actions of the hormone without regard to which one is primary-they are both obviously important. Unlike vitamin D, the hormone has little effect on the absorption of either calcium or phosphorus in the gut. Overdosage with parathyroid hormone brings about bone demineralization and hypercalcemia.
Hypercalcification, on the other hand, may result from massive doses of vitamin D. The hormone has no effect on the healing of rachitic bones, but in a hormone deficiency tetany vitamin D is effective in regulating the blood calcium and phosphorus levels. Usually large doses, 50,000 to 200,000 IU of vitamin D per day, are required for this in humans. Another important drug in this respect is dihydrotachysterol (AT 10). This product has been used in human parathyroid hormone deficiency with considerable success.
It is administered orally in oil solution, a distinct advantage over parathyroid hormone, which must be given parenterally. AT 10 is very slightly antirachitic; it increases calcium absorption from the intestine, brings about resorption of calcium from bone to increase the blood level, and increases urinary excretion of phosphate. The last two actions are those of the parathyroid hormone; the first two are those of vitamin D. Quantitatively the actions of AT 10 are less than those of either the vitamin or the hormone. Albright and co-workers have studied the effects of the three substances, parathyroid hormone, AT 10, and vitamin D, important in the physiological regulation of deranged calcium and phosphorus metabolism in a variety of clinical conditions.
Calcium Urinary Phosphours
Absorption Excretion
Vitamin D ++++ ++
Dihydrotachysterol ++ +++
Parathyroid hormone + ++++
Vitamin D, especially in large doses, is somewhat effective in bringing about calcium resorption from bone; AT 10 is far more effective; and the parathyroid hormone has still greater activity. The hormone has a very short action, a matter of hours, and may bring about its C3Xlffium effect very rapidly (four to ten hours). AT 10 has a longer period of action, up to 'eral days, and requires, a day or two to act.
The vitamin may be effective for long periods of timeand is slow to exert its effects. Mutiple C14 labeled calciferol was prepared by Kodicek. One mg of the compound in oil was given to a rachitic rat by mouth. After 24 hours, analyses showed that only 30 per cent of the C14 activity
remained as vitamin D. although all the CH activity was recovered. The bulk of the breakdown products were not identified. The liver contained 5.7 per cent of the dose, the bones 1.4 percent, i.e intestines 0.6 per cent, the kidney 0.2 per cent, and the blood 1.2 per Cent. It was of :nterest that tissues concerned with phosphate turnover. such as kidney. bone and intestine. contained significant amounts of the labeled vitamin D.
