Quarterly Technical Report April 1 Thru June 30, 1953

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Health and Biology THE urm7JERSITY OF ROCHESTER Atomic Energy Project Rochester 20, New York QUARTERLY TECHNICAL REPORT April 1 thru June 30, 1953 It should be noted t h a t the Q,uarterly Technical Reports of The University of Rochester Atomic Energy Project do not attempt t o describe progress i n all of the research programs but only i n those i n which some significant results have been achieved but which are not sufficiently cmplete t o be written up as final reports. Submitted by: Henry A. Blair Date of Report: 9/2/53

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Selected Studies on the Role of Bone Phosphatase i n A Statistical Evaluation of Certain Factors Involved i n the Plating of Polonium by Frank A. Smith and Rocco The Effect of a Soft Diet on Radiation Illness in the Effects of a Minimal Lethal X-ray Exposure of Sensitization of .Mice t o Histamine by William K. Cotton and Robert W. ABSTRACTS OF PAPERS PRESEmTED AT MEETRJGS Phosphatases of the Cell-Surface of Intestinal Cells by Age Increase i n Fluoride Content i n Human Bone by Frank A. Analytical Chemistry of Micro-Quantities of Beryllium Determination of Calcium in Biological Material by Flame The Inhibition of Glucose Uptake and of Respiration of Hemin Synthesis in Polonium-Induced Anemia i n the Rat by Robert G. Tnomas, K u r t I. Altman, J. Newell Stannard and Metabolism of Adrenal Cortical Hormones i n the Isolated Chemopathology of Hemoglobin Synthesis in Mice with Hereditary Anemia by Kurt I. Altman, Elizabeth R . Russell, Kurt Salmon and James K. Scott. ..............

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ABSTRACTS OF PAPERS PRESENTED AT MEETINGS (Cont.) An Heptic Factor Essential for Maximal Synthesis of Protein by the Walker Tumor in the Rat by Leon L. Miller, James A. Fancher and William F. Bale ................. 22 Maximum Permissible Amounts of Radioisotopes in the Human Body and Maximum Permissible Concentrations in Air and Water

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SEUCTED STUDIES ON THE ROLE OF BONE PHOSPHATASE IN CALClFICATION A s p a r t of the general study of the problem of calcification, special attention has been given the role of phosphatase which was first ascribed a major r o l e by Robison. A number of experiments have been performed with the aim of defining more precisely the specific part i n the calcification process played by phosphatase. Because the Robison theory of calcification is based on a nonexistent solubility product for the bone salt, this theory i s not tenable. . Robison's work, however, i s of lasting importance because he drew attention t o the possible part phosphatase may play i n calcification. The physiological role of phosphatase i n calcification seems t o be atleast twofold: of the bone matrix and (b) it protects the calcification process from (a) it may contribute i n some way t o the formation the inhibitory effect of ester phosphate. Attempts t o explain why ester phosphate exerts a beneficial effect on calcification have not been fruitful in giving a new theory of calci - f ication but considerable clarification of the problem has been achieved. It has been sham: respiration of ra chiti c cartilage. (a) That ester phosphate does not contribute t o the It i s not likely that phosphate esters maintain the calcifying mechanism by an effect on respiration. (b) That ester phosphate does not diffuse through a cellophane membrane at a faster rate than inorganic phosphate. ester phosphate and inorganic phosphate on calcification in vitro is probably not due t o differences in the rate of diffusion of the two The difference in the effects noted with ( c ) That phosphatase does not act as a transphosphorylase between

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the bone matrix and glycerophosphate. not contribute phosphate for the transphosphorylation of the hypothetical 'zlhis means that glycerophosphate does collagen fiber template. (d ) That bone phosphatase does not remove phosphate ions fromthe site of cslcification. i s not of a critical nature. investigation a t the time of this writing. attractive and i f proven would elucidate the mechanism whereby ester phosphate aids calcification. Reasons are given why this experiment mew approaches to this problem are under It has also been demonstrated that glycerophosphate i s hydrolyzed comparatively rapidly by phosphatase. that glycolytic inhibitors do not affect calcification --i n vitro when glycerophosphate is the source of phosphorus but are paterful inhibitors of calcification when biological esters are used. shown t h a t glycolyLic inhibitors are effective in inhibiting calcification because. of interference with cellular metabolism. a preformed surface, upon which mineral deposition takes place, deteriorates in the absence of cellular activity. Degeneration of the surface or template distorts the spatial arrangement of the phosphate groups there-. This inbibits the formation of the bone salt lattice. This sheds some light on the reason Further exploration has Tt is suggested that ...AT P is not beneficial t o calcification of ra chiti c cartilage from the rat i n v i t r o because (a) it is not attacked by the bone enzyme and only slightly by enzymes present i n the slice and (b) it forms only slightly dissociated complexes with calcium ion.

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A STATISTICAL EVALUATION OF CERTAIN FACTORS Frank A . Smith and Rocco Della Rosa Factor idly designed experiments have been completed i n which the ’ influence of a number of factors involved i n the spontaneous plating of polonium from acid solutions were measured. From the results obtained, plating conditions were found such t h a t the standard deviation of a single determination is five per cent of the activity present. The experiments t o be described were undertaken i n order t o furnish a reliable estimate of the influence of a number of conditions operative i n the spontaneous plating of polonium from acid solutions. The first experiment was designed t o measure the effects on plating efficiency of temperature, time, volume of solution, stirring, acidity, and evaporation. Each of the six factors was tested a t two levels in a factorial - l y designed experiment. The quantity of polonium was kept constant and w a s equivalent t o 4000 counts per minute i n each smple. The factors and levels tested are listed in Table 1. A total of 32 platings were done. All foils were counted i n scintillation counters i n such a manner that the counting error did not exceed +- 2.5 per cent. The influence of each factor on plating efficiency was assessed by a statistical analysis of the counts obtained. he factorial experiments required were designed and analyzed by Drs. S. Lee Crump and A.M. Dutton of the Statistics Division. acknowledge our indebtedness for their assistance and cooperation. We are pleased t o

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Factors Studied Factorially for Their Influence on the Sgontaneous Plating of Polonium from Acid Solutions Temperature of plating bath 45°C and 90°C Time of plating 45 min. and 90 min. Volume of plated solution 50 ml ana l o o ml Acidity of plated solution as slow and as fast as practicable Evaporation of plated solution and not replenished' vol. replenished frequently 'When not replenished, approximately half of the volume w a s lost by evaporation. The data resulting indicated that the rate of stirring, and loss of volume by evaporation did not affect significantly the quantity of polonium plated on the foil. Efficiency of the plating process was improved a t the higher plating temperature, longer plating times, and i n the larger volume of plating solution. The efficiency also was somewhat greater i n the less acid solutions, though the improvement was quite s m a l l . It was concluded that the effects of time, temperature, initial volume, and evaporation need not be investigated further. solution, and of stirring appeared t o warrant further study. prove unnecessary, the design of the plating apparatus obviously would be The effects of lesser acidity of the plating

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Accordingly, a second factorial experiment was prepared t o study further the effects of these factors. Three concentrations of acid were used, namely 0.1, 0.33 and 0.75N. Stirring was done a t as rapid arate as practicable, or eliminated entirely. The level of activity of polonium was 200 counts per minute. counts, and a third time at a level of 200,000 counts per minute. The experiment w a s repeated again, using 20,000 of 72 platings were made. ... The results obtained indicated again that somewhat better plating resulted at the lower normality of acid. Stirring of the solution definitely is required since in stirred solutior?s the quantity of polonium spontaneously plated waB approximately two times The improvement was slight, however. greater than was obtained i n unstirred samples. Using the IO5 observations made, the standard deviation was calculated t o be five per cent of the activity present for the range 200 - 200,000 counts per minute. The standard deviation vazied directly with the amount of A detailed report of these experiments, prepared i n collaboratlon with the Statistics Divlsion, will be issued later.

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TaE EFFEET OF A S3FT DIET ON RADIATION ILLNESS IN THE DOG A Goft diet fed to dogs before and after an LD/50 exposure to x-ray was ineffective in reducing the degree or extent of gastro-intestinal hemorrhage. It has been suggested that a soft, low residue diet may alleviate the gastro-intestinal hemorrhage of acute radiation sickness. milk, non-nutritive cellulose fiber and Abbott's vitamin B elixir with crude liver was used. Ten dogs received this diet for one moth prior to rahiatiun asd awing the post-radiation period. A control group of 10 dogs received Purim dog chw kibbled mealo The dogs received 450 r of 250 KV x-irradiation (19/50)o S i x of the dogs 0;1 the soft diet and 3 of the control dogs died. At necropsy, one o f tb.e control dogs shared no gastro-intestinal bleeding. A l l of tBe other dogs that. died shared mild to moderate Bmall and Large bowel hemorAt this dose of radiation, this soft, l c c w residue diet had no effect on diminishing t b e degree or extent of' gastro-intestinal hemarrhage.

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ON SENSITIZATION OF MICE To HISTAMINE William K. Cotton and Robert W. Miller From the results obtained, an exposure to x-irradiation resulting in a mortality of between an LD/lO-LD/l5 apparently does not,sensitize mice to histamine in dosage levels of either 50 mgm./kg. or 1000 mgm./kg. given between It has been sham by various workers that injections of sub-lethal doses of H. Pertussis vaccine into mice sensitizes these animals to relatively small amounts of histamine (1, 2, 3 ) . Kind (2) suggests that the vaccine injected may produce adrenal cortical damage and thus Interrupt the normal mechanism of response to stress. Parfentjev and Goodline (3) were able to demonstrate that 5 days after injection of H. Pertussis vaccine, 2 mg. of histamine diphosphate was as toxic to the sensitized mice as .5O mg. was to pormal animals. To date, the effects of ionizing irradiation upon the a d r e d cortex is Brayer, Ghsser and Duffy (4) have sham a temporary elevation in the excretion of adrenal. steroids following x-ray exposure in pigs. Little is known,harever, concerning the production of adrenal steroids following irradiation. Apparently, the adrenals are relatively radio-resistant. This experiment was devised to determine the effects of a plsximal T;D/O exposure of x-irradiation on the sensitization of mice to injections of

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1. Mice - 210 female, all purpose, Albino mice from the Rockland Farms, 3. X-ray factors - 350 r whole body x-irradlation from a 250 KV Picker Urn. parabouc 1/2 mm. Cu. Ten mice were injected with lo00 mg./kg. of histamine diphosphate I.P. ana served as the histamine control groq. body x-irradiation and served as radiation controls. were irradiated as above and in addition received injections of histamine as follovs: eighty out of the one hundred and sixty in groups of twenty mice each, were injected with 1wO mg./kg. of histamine diphosphate on the 5th, loth, received 50 mg./kg. of histamine diphosphate on the EWIE schedule. 8nimJ.s used in this experiment were groqed by randam selection. Forty mice received 350 r of whole One hundred and sixty mice The other eighty mice, in groups of twenty, It was expected that the above dosage of x-ray would result in an XD/O martality and therefore death of the nninwlfl following injection with histamine could serve as an index of sensitization following a sub-lethal exposure to these animals having died wlthaut receiving histamine; therefore, the mortality However, the mortality in the group was 29/2OO; 19 of tween 1-15 days after receiving their injections. It would be expected that i-fl the deaths were due to sensitization to histamine, they would have o c c m e d vitbin 24-48hours after the administration of the histamine.

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Frau the results obtained, an exposure to x-irradiation resulting in a mortality of between an I;p/lO-LD/l5 apparently does not sensitize mice to histamine i n dosage levels of either 50 mgm./kg. or 1000 mgm./kg. given between 1. Smith, W. S.: Acute KC1 and Histamine Tolerance and Adrenal Weight i n on Adrenal Steroid Ekcretion in Pigs, University of Rochester Atomic Energy Project Quart. Tech. Report, UR-260 p. 12, 1953.

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Abstract of Paper Presented at Chicago, Illinois April 6-io1 1953 There was little or no direct absoqtion of glucose-1-3hosphate from the intestine of the rat. However, the sugar phosphate and various other phoqhate compounds were rapidly hydrolyzed in intestinal loops. One of the products, orthophosphate, could be almost completely recovered i n the lumenal content 8 . The second product, glucose, was only partially recovered because of absorption of this substance from the intestinal lumen. The rate of hydrolysis of glucose-1-phosphate was about the same i n - all parts of the small intestine and also was the same i n excised loops as i n " i n vivo" loopse I n contrast the absorption of glucose was variable along the length of the intestine, and was markedly reduced in excised loops. When glucose-1-phosphate with P32 incorporated was hydrolyzed, there was no equilibration of labeled phosphate with the phosphate of the intestinal cells, indicating that the hydrolysis was _not taking place i n the interior of the c e l l s . Nor was any phosphatase secreted into the lumen under the conditions of these experiments. Therefore it i s concluded that the phosphatases concerned are bound on the surfaces of the intestinal cells.

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&st rac t of Paper of the Federation Presented at the Thirty-Seventh Annual Meeting of bri can Societies for Experimental Biology Am INCREASE Is FLUORIDE CoIlTENT IX HUMAN BCEIE Frank A. Smith, Dwight E. Ctardner and Harold C. Hodge It has been well established that fluoride retained i n the animal body i s deposited almost entirely in the skeletal system. have been made on human bone. analyzed indicate the need fora more solidly substantiated baeis for the However, the much smaller number of samples normal bone fluoride concentration, in order to offer a reliable …