Henry L. Jaffe

Phosphatase Studies. IV. Serum Phosphatase of Non-osseous Origin. Significance of the Variations of Serum Phosphatase in Jaundice

In view of the wide occurrence of phosphatase in other tissues than bone, the possibility of non-osseous origin of some of the serum phosphatase deserves examination. It receives support from the evidence of the great decrease of serum phosphatase after prolonged fasting 1 and its great increase after ingestion of dextrin. 2 The significance of phenomena of normal physiology has frequently been suggested by a disturbance of the function of a given organ or tissue; we believe that the increase of serum phosphatase in jaundice is of similar importance and indicates that liver is a source of serum phosphatase, normally as well as pathologically. The procedure for determination of serum phosphatase has been described.3∗ The icteric index was determined by Meulengrachts method. Results. Serum phosphatase was increased in about 50 observed cases of jaundice, with the exception of some cases of anemia. In acute catarrhal jaundice bile pigments elaborated in the liver, and phosphatase, presumably of the same origin, were found in the serum in increased quantities. Yielding readily to treatment, this condition was particularly suitable for a demonstration of the decline of the serum phosphatase from its high initial values (Table I). Other clinical conditions associated with jaundice and high serum phosphatase were not equally suitable for a study from the physiological standpoint. The decrease of the icteric index during treatment corresponded with the clinical improvement. The figures in Table I show that serum phosphatase decreased with the icteric index. It may be noted in Case 8 that not only did serum phosphatase decrease as the case improved, but that it increased during relapse.

Effects of Diet and Fasting on Plasma Phosphatase

Summary and Conclusions Rats maintained for over a month on the Sherman diet showed a lower plasma phosphatase than those maintained on a meat diet. The plasma of fasted rats and guinea pigs had a much lower phosphatase content than the plasma of fed controls. The variations of plasma phosphatase could not in these cases be attributed to any phenomena associated with the physical and chemical processes taking place in bone. Indeed, the assumption that plasma phosphatase changes are caused only by physical or chemical changes in the bone would lead to expect results contrary to those we actually obtained, because starvation for 3 to 6 days causes considerable non-specific bone resorption. 7 It seemed more plausible that the physical and chemical processes associated with nutrition were closely linked with these changes in plasma phosphatase. It is possible, however, that the cessation of new bone formation in young animals (also observed during starvation) contributes to the lowering of plasma phosphatase. It must be noted in this connection that young, growing animals were used, and that our conclusions do not necessarily apply to adults.

Phosphatase Studies. VIII. Increase of Serum Phosphatase After Bile Duct Ligation in Dog

Roberts has shown increased plasma phosphatase in obstructive jaundice. 1 ,2 We have demonstrated an increase of serum phosphatase in a series of cases of catarrhal jaundice, and a return to normal values after their clinical improvement. 3 We interpreted these data as supporting the assumption of a hepatogenic source of serum phosphatase. It seemed desirable to study the subject experimentally—by bile duct ligation in one group of experiments. The operation was performed under amytal anesthesia upon a young adult female dog weighing 11.6 kilos. The dog made a good recovery. Observations covered a period of over 7 weeks. About 4 weeks after the operation, the dog having had repeated attacks of vomiting, it was decided to discontinue food for a few days and then to resume feeding with certain precautions. Advantage was taken of this period of fasting in order to compare the results with earlier observations on the effects of fasting on normal dogs. 4 At the time of the last analysis the inorganic phosphorus determination indicated impairment of kidney function. This indication was supported by the findings of 51.7 mg. of urea N per 100 cc. of blood and of 2.1 mg. of creatinine. At the same time the uric acid content of the blood was 4.8 mg. per 100 cc.; in dogs with normal liver function the uric acid content of blood is too low to be determined. The dog was killed by administration of ether. Immediate necropsy revealed multiple areas of liver necrosis.

Functioning autoplastic suprarenal transplants.

No proof has as yet been presented that free transplants of the suprarenal gland function. Our experiments, with the view of obtaining evidence of function, were carried out on both the rat and the guinea pig. We realize the difficulties of interpreting results in favor of function in the rat, particularly if the clinical condition of the animal is taken as an index, because of the frequent occurrence of accessory suprarenal tissue. For crucial evidence of functioning free transplants we used the guinea pig because continued survival of this species after complete bilateral suprarenal ablation has never been reported. The best results are Rogoffs, 1 who, in a series of 17 animals with an interval of 2 to 3 weeks between the removal of the right and left glands, reports that 1 pig lived 28 days, while 14 of the 17 died during the first 8 days. PROCEDURE. In the rat both glands were removed in one sitting and placed in sterile physiological saline at about 39° C. Each gland was cut in half and the four parts were immediately transplanted into pockets between the fascia and abdominal muscle, or in the abdominal muscle. In 3 or 4 weeks the transplants regenerate as highly vascularized masses of cortical tissue, with the three cortical layers. Medullary tissue does not regenerate. Transplants in rats sometimes attain the size of suprarenal glands of adult animals. In the guinea pig difficulties are encountered in transplantation because the muscle of this animal is highly sensitive to epinephrin, as described by Elliott and Tuckett. 2 Small amounts of this drug when introduced with the transplant may cause marked swelling, and this may he followed by sero-sanguinous oedetna, necrosis, sloughing, and infection of the abdominal wall, so that the transplants are destroyed.

Production in Guinea Pigs of Fibrous Bone Lesions with Parathyroid Extract

Askanazy 1 found a parathyroid adenoma in a case of ostitis fibrosa and he suggested a cause and effect relationship between the parathyroid tumor and the bone disease. After his finding, many other instances of parathyroid enlargement were reported in association with ostitis fibrosa deformans, ostitis fibrosa cystica, osteomalacia and rickets. Parathyroid enlargement has also been noted in rats suffering from experimental rickets. The consensus until recently was that the parathyroid enlargement observed in association with these bone diseases was of a secondary nature, and appeared as a result of a compensatory hypertrophy due to the bone deficiency. Mandl, 2 and after him others, removed parathyroid adenomas in cases of ostitis fibrosa cystica and reported rapid clinical improvement of their patients, with cessation of the negative mineral balance. We felt that, if the extirpation of a parathyroid adenoma resulted in the clinical improvement of a case of ostitis fibrosa cystica, injections of parathyroid extract might produce similar or analogous bone lesions, if parathyroid hypersecretion was at the basis of the disease. Attempts have been made to produce experimental ostitis fibrosa by dietary deficiencies and by injury to the bone marrow 3 , 4 with negative results. Dogs treated with parathyroid extract develop very extensive bone resorption, in both the cortex and the medulla, but fibrous repair is difficult to elicit. It is suggestive that in the dog doses large enough to raise serum calcium to very high levels and to produce a recognizable overdosage complex, sometimes leading to death, are not sufficient to cause lesions severe enough to elicit the response of fibrous repair. In the guinea pig, however, doses which, while high in terms of parathormone units, produce very much smaller effects on serum calcium and few, if any, of the overdosage effects, will cause bone lesions with fibrous repair.

Experimental Ostitis Fibrosa Cystica in Dogs.

In previous reports the production in the guinea pig of fibrous bone lesions by injection of parathyroid extract was described, as well as its effects on the serum calcium and phosphorus. 1 , 2 The production in dogs of ostitis fibrosa cystica is possible, but more difficult. The difficulty lies in the fact that in the dog doses of parathyroid extract (Parathormone Collip) necessary to produce marked resorption of the bone and marrow injury, are liable to lead to fatal hypercalcemia before there is much fibrous repair. We studied eleven growing puppies for periods from 10 to approximately 180 days. They were under the influence of increasing doses of hypercalcemia parathormone. We produced, depending upon the dosage and the length of time under parathormone, all degrees of change from mild bone resorption and slight fibrous replacement of the marrow to severe bone resorption and degeneration of the marrow with hemorrhage, when the animals died from overdosage. Finally, we produced typical ostitis fibrosa cystica in 3 dogs that were injected for 5 to 6 months. In these we gradually increased the daily dose of parathormone. At the end of the experimental period they were receiving 20 units daily. The dogs with the more pronounced lesions showed: (1) resorption of the existing spongy and cortical bone, (2) invasion of the enlarged haversian spaces and of the marrow canal by fibrous tissue, (3) the presence of Howships lacunae, containing osteoclasts, on the walls of the haversian spaces, on the inner and outer surfaces of the compacta, and on the surfaces of the spongy trabeculae, (4) new bone formation (osteoid tissue), as a substitute for the original lamellar bone, and (5) cysts and hemorrhages in the marrow cavity.

Serum phosphatase changes in calcium deficiency and in ammonium chloride osteoporosis.

It was shown by Kay 1 that the plasma phosphatase increases in certain clinically observed cases of bone diseases. We have confirmed and extended these observations. 2 In an investigation of experimentally produced bone lesions we have reported the effects of chronic and acute hyperparathyroidism. 3 In view of the many analogies observed in bone resorption, whether produced by parathormone or by other agents, it was desirable to extend our observations to the osteoporoses produced by low-calcium diets and by ammonium chloride administration to animals on low and high calcium diets. Four litters of dogs were used—3, 6, 8, and 18 months old, respectively. The experiment was continued for about 11 weeks. The animals received a diet of fresh, lean horse meat supplemented with cod liver oil and tomato juice. This is a low-calcium diet. Some of the animals received a calcium supplement (2.5 gm. each of bone meal and calcium lactate per kilo of food) equivalent to between 0.5 and 2.5 gm. of calcium daily. On this diet the controls grew rapidly; the bones were normal upon autopsy. On the low-calcium diet, the growth was equally rapid; the bones, however, were thinned. Ammonium chloride was administered by stomach tube in a 1% solution. At the end of the experiment some dogs received daily as much as 1 gm. per kilo. In the youngest litter bone softening and deformity resulted on a low-calcium diet; in all dogs administration of ammonium chloride generally caused loss of appetite. The method for determination of phosphatase has been described. 4 , 5 Serum phosphatase rather than plasma phosphatase has been determined in this series of tests to avoid the inhibition of phosphatase activity by oxalate. 5

Experimental studies on the formation of Hassall's corpuscles.

Conclusions 1. These studies seem to bring unequivocal experimental proof that Hassalls corpuscles are derivatives of the reticular epithelium; a view originally proposed by Paulitsky and more recently elaborated by Hammar, and supported by many others on the basis of embryological and post fetal histological studies. 2. They also show that in post fetal life the formation of Hassalls corpuscles is- independent of the presence of remnants of the original epithelial ducts of Remak.

Serum Calcium and Phosphorus of Guinea Pigs after Administration of Single and Repeated Doses of Parathormone

The tendency has been to assume that hyperparathyroidism is consistently associated with hypercalcemia. We emphasize that frequently this is not the case, that hypercalcemia is not a sole criterion of hyperparathyroidism. Recorded instances of absence of hypercalcemia after parathyroid administration have sometimes been noted as merely paradoxical. The suggestion of “immunity” to parathormone is not supported by the data. On the other hand, the suggestion that an increased rate of excretion may prevent the accumulation of calcium in the blood is plausible. And a generalization (based on work with rodents) that herbivora are very resistant to parathormone injections implies an effect of diet on serum calcium response. However, the resistance of the cat would have to be explained on a different basis. It has been established that in mice, rats and rabbits relatively small, if any, effects upon serum calcium are produced by administration of relatively huge doses of parathormone. Negative results in guinea pigs were reported by Macleod and Taylor 1 and Taylor. 2 We found that in guinea pigs consistent effects upon serum calcium and phosphorus could be produced only by very large doses; that these effects could be brought out more prominently, in young animals, after starvation; and that with doses not sufficiently great to produce effects on serum calcium and phosphorus, calcium mobilization and excretion could be demonstrated. Controls. Normal guinea pigs, young and adult, fed and starved, showed a serum calcium of 10.4±1.0 mg. per 100 cc. The serum phosphorus varied from about 8.0 mg. in guinea pigs weighing about 300 gm. to about 4.0 mg. in adults. Starvation for about 70 hours lowered the serum phosphorus of the young animals to between 5.0 and 6.5 mg. Effects of a Single Administration of a Large Dose: I. Calcium. With doses of 10 to 20 units per 100 gm., injected subcutaneously into adult guinea pigs, the serum calcium rose to a maximum of about 16.0 mg. about 24 hours after injection and returned within the normal range before 48 hours. Starvation did not intensify the effect.

Experimental Chronic Hyperparathyroidism in Dogs Without Hypercalcemia.

The rate at which the elimination of tissue and bone calcium proceeds in the dog under the influence of parathormone depends, among other factors, upon the dosage. However, the dose of parathormone that may be administered to the dog is limited by the danger of fatal hypercalcemia. Serum calcium is obviously one term in an equilibrium. Some of the other terms affecting it, under given physico-chemical conditions of the blood, are: tissue calcium and other salts, bone salts, their availability, and the rate of calcium excretion, particularly by the kidneys. The rapid rate of excretion of calcium, as well as dietary factors, are probably largely responsible for the phenomena observed by us in the guinea pigs after parathormone. We have shown 1 , 2 that this animal tolerates large doses of parathormone, although it reacts to the extract both by an elevation of serum calcium and by formation of bone lesions. In our belief, this “tolerance” is the reason for the ease with which the typical changes of ostitis fibrosa were produced in guinea pigs. We have succeeded, after having depleted bone and tissue calcium in dogs, in administering to them relatively large doses of parathormone without producing hypercalcemia. By thus increasing the “tolerance” of the dog to parathormone it was possible to produce ostitis fibrosa cystica in this animal. 3 This report is based on a study of 11 growing dogs in which experimental chronic hyperparathyroidism was produced by daily injections of parathormone. The diet of the dogs consisted of an adequate quantity of fresh raw meat with additions of cod-liver oil, bone meal, calcium lactate and tomato juice. Parathormone was injected at first in a daily dose of 2 units per kilo, which was raised gradually in some dogs to as high as 5 units toward the end of the experimental period of 5 to 6 months.