Aaron Bodansky

Determination of Plasma Phosphatase.

The significance of phosphatase in bone physiology was suggested by Robison and his coworkers. Increased plasma phosphatase was recently reported by Kay in ostitis fibrosa, ostitis deformans, active rickets and osteomalacia. Kay also reported a method for the determination of plasma phosphatase. 1 In an experimental study of plasma phosphatase in ostitis fibrosa, which we produced in young guinea pigs and dogs by prolonged treatment with parathormone 2 , 3 , 4 , 5 we found Kays method of determination of plasma phosphatase unsatisfactory. We have avoided the disadvantages of Kays method by the use of an M/10 solution of sodium diethyl-barbiturate (veronal) as a buffer.∗ When it is added to plasma in the proportions which we employ, the resulting mixture has a pH of about 8.6. This reaction is not changed after the addition of the substrate—a solution of sodium beta-glycerophosphate (Boots Drug Company, Nottingham, England) containing about 5 mg. of phosphorus per cc.† ‡ Nor does the pH change perceptibly during the action of the phosphatase, even when continued for 72 hours.

Rate of decalcification and the sites of bone lesions in experimental hyperparathyroidism.

A relatively rapid rate of mineral metabolism is associated with rapid bone growth in young animals. Our studies in acute and chronic experimental hyperthyroidism have shown a greater susceptibility to parathormone and a more rapid excretion of calcium in the young than in the adult. 1 , 2 Bauer, Aub and Albright 3 stated that in bone resorption the calcium of the trabeculae is “labile” and is drawn upon in the first instance, while the calcium of the compact bone (“the structural part of the bone”) becomes available only “in the case of unusual body demands.” Our studies suggest another conception: If we are to speak of labile calcium, it is the calcium in the regions of most active growth; if we are to speak of less readily available calcium, it is the calcium in the regions of less active growth. We have examined the skeletons of about 150 young guinea pigs, 50 old guinea pigs, 25 young dogs, and 10 rats. The so-called characteristic lesions were severest and most easily produced in young animals suffering from acute and chronic hyperparathyroidism. The bones and portions of bones most affected were the metaphyses of the long tubular bones, and the cortex of the shaft, particularly near the epiphyseal cartilage plates; the costochondral junctions, and the cortex of the ribs used most in the respiratory act; the bones of the skull and of the lower jaw. The metaphyses of the slower growing short tubular bones showed relatively few lesions; the cortex of the shafts of these bones showed practically none. In the guinea pig, most epiphyses are already formed and the centers of ossification for the tarsal and carpal bones are present at birth. No specific lesions were found in these bones, at most a simple atrophy of their trabeculae was observed.

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.

Antagonistic effects of insulin and thyroxin

Continued subcutaneous administration of thyroxin is known to produce hyperglycemia. In normal sheep the blood-sugar is raised from about 70 mg. to over 80 mg. A single subcutaneous administration of thyroxin has only a slight effect on the blood sugar. A single subcutaneous administration of insulin (10 units) produced no demonstrable effect within two hours after injection. Intravenous administration of varying amounts of insulin (5 to 15 units) produced prompt hypoglycemia in normal sheep, the sugar decreasing from about 70 to about 40 mgs. per 100 c.c. of blood within about 30 minutes. (No unfavorable symptoms were observed). The drop is regular, and when plotted is represented by a practically straight line. The slope of this portion of the curve is constant for the same sheep upon administration of varying amounts of insulin. The portion of the curve representing recovery from hypoglycemia show characteristic differences : When 5 units of insulin were administered, the recovery to normal sugar values began immediately after the low values had been reached. With 10 to 15 units the minimum sugar values obtained were only slightly lower than for the smaller doses, although analyses were taken at sufficiently close intervals (10 to 20 minutes) to enable a fair approximation to the minimum. The larger doses, however, showed an extensive flat portion in the curves before recovery began. The results with five units were reproducible with the same sheep and similar in different sheep. Five units were therefore selected as the standard dose of insulin to be tested against varying amounts of thyroxin under different conditions. Administered alone, the effects produced are represented by curves A and D. Curve G shows the effect of a preliminary administration of thyroxin (1 mg. intravenously). Curve C shows the effect of a similar injection of thyroxin after the recovery from insulin had begun. The simultaneous administration of thyroxin (1.5 mg.) and insulin produced a typical divergence of the ascending arm of curve E from that of curve D (insulin alone)

Paradoxical increase of phosphatase activity in preserved serum.

In the course of examination of the liability of serum phosphatase several samples of serum were saved for 24 hours under ordinary laboratory conditions (for about one-half to 1 hour at room temperature and in the refrigerator thereafter). It was expected that the phosphatase values would be similar to or lower than the values In the course of examination of the liability of serum phosphatase found in fresh serum. The following data, typical of results obtained in about 30 different comparisons, will show unexpectedly increased values. In these tests the differences between the 24-hour serum and the fresh serum phosphatase activities were greater than discrepancies usually found in determinations performed simultaneously upon 2 samples of the same serum (about 2%, rarely as high as 5%), and they were all in the same direction. It may be noted that the inorganic phosphorus in the preserved serum remains unchanged. The interest of these results lies in their unexpectedness. Under the conditions of the test 1 , 2 the assumption of an increased pH causing increased phosphatase activity is excluded (1) because our buffer maintains a pH constant during hydrolysis at about 8.7 and (2) because any slight increase in the pH that might occur with the preserved serum could not cause increases of phosphatase activity as great as those observed. It is possible (1) that during the 24 hours intervening between the separation of the serum and the analysis some factor inhibiting phosphatase activity is destroyed; (2) that a component of serum phosphatase is activated; or (3) that substances are formed which further phosphatase activity. However, the mechanism and significance of this apparent increase in phosphatase activity of preserved serum remain to be elucidated.

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.

Effect of thyroidectomy upon the reaction of sheep to insulin

The author has suggested previously that the effect of thyroxin upon glycogenolysis could be demonstrated after the synthesis of glycogen brought about by the administration of insulin, and submitted some experimental evidence. 1 Further evidence of the glycogenolytic function of the thyroid is yielded by data upon the reaction of normal and thyroidectomized sheep to the intravenous administration of a standard dose of insulin. In normal sheep a regular drop in the sugar content of the blood takes place, the sugar values reaching a minimum within about 35 minutes after the injection. Recovery to normal sugar values begins immediately, being complete within 1 1/2 to 2 hours after the injection. In thyroidectomized sheep the decrease of the sugar values continues until about 50 minutes after the injection and a more intense hypoglycemia is produced. Recovery to the initial sugar values proceeds at a strikingly slower rate than in normal sheep. It is suggested that the thyroid is essential to normal recovery from insulin hypoglycemia. Experiments are proceeding upon clinical applications of this principle. This work was carried out under a grant from the Heckscher Research Foundation.

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.