REQUEST FOR APPROVAL FOR HUMAN USE OF RADIOISOTOPES IN TRACER AMOUNTS IN VOLUNTEER EXPERIMENTAL RESEARCH SUBJECTS Submitted by: U.S. Army Medical Research and Nutrition Laboratory Denver, Colorado M.E. McDowell, M.D., Lt. Col., MC, Commanding Officer and Director J.E. Canham, M.D., Lt. Col., MC; Chief, Metabolic Division J.E. Hansen, M.D., Lt Col., MC; Chief, Physiology Division E.M. Baker, Ph.D., Maj., MSC; Assistant Chief, Chemistry Division and Chief, Carbohydrate Branch K.E. Kinnamon, DVM, Capt., VC; Chief, Radioisotope Branch, Physiology Division J.R. Handy, M.D., Capt., MC; Chief, Cellular Physiology Section, Physiology Division H.E. Sauberlich, Ph.D., (PL-313), Chief, Chemistry Division G.A. Leveille, Ph.D., (GS-13), Chief, Lipid and Protein Chemistry Branch, Chemistry Division B.M. Tolbert, Ph.D., Professor of Chemistry, University of Colorado and Consultant to USAMRNL AND Fitzsimons General Hospital Denver, Colorado C.A. Moore, M.D., Lt. Col., MC, Chief, Urology Service O.G. Stonington, M.D., Professor of Urology, University of Colorado School of Medicine and Consultant to Fitzsimons General Hospital Section I. General Introduction Paragraph Purpose of request 1 Scope of request 2 General 3 History of USAMRNL isotope usage 4 Specific radioisotopes to be used 5 Section II. General Health Physics for Requested Isotopes* Carbon-14 6 Hydrogen-3 7 Magnesium-28 8 Calcium-47 9 Calcium-45 10 * (for references consult Appendix I.) Section III. Proposed Nutrition and and Metabolism Tracer Studies Vitamins 11 Amino Acids 12 Lipids 13 Carbohydrates 14 Minerals 15 i Appendices I. References on General Health Physics Appendices II. Voluntary Consent Statement Appendices III.RDTE Facilities Fact Sheet, USAMRNL, 18 November 1963 ii Section I. General Introduction 1. Purpose of request a. Par. 3b(3), AR 40-37, "Radioisotope License Program (Human Use)," dated 12 August 1963, requires that written approval to be obtained from the Secretary of the Army prior to the submission of license application (through channels to AEC) for human use (of radioisotopes), when volunteers are to be used as experimental research subjects. This paragraph (Par. 3b(3) AR 40-37) cites AR 70-25, "Research and Development: Use of Volunteers as Subjects of Research," dated 26 March 1962 as the basis for the requirement. b. AR 70-25 prescribes policies and procedures governing the use of volunteers as subjects, including research in nuclear, biological and chemical warfare, wherein human beings are deliberately exposed to unusual or potentially hazardous conditions. Par. 6 of this AR requires approval of the Chief of Research and Development prior to the research and, in the case of nuclear, biological or chemical agents, approval of the Secretary of the Army is required. c. The Atomic Energy Commission will license the use of tracer amounts of radioisotopes in physiological studies in normal human beings done by competent medical research scientists. Such licenses have been granted to members of this organization in the past. 1 d. To comply with the requirements of Par. 3b(3), AR 40-37 and Par. 6, AR 70-25, this request is submitted for approval for human use of stated radioisotopes in tracer amounts in volunteer experimental research subjects at U.S. Army Medical Research and Nutrition Laboratory and Fitzsimons General Hospital an in field studies conducted by USAMRNL. 2. Scope of request a. Experiments included in this request are not, in and of themselves, unusual or potentially hazardous under the definitions of AR 70-25. They would be considered potentially hazardous (and minimally so) only to the extend that radioactive isotopes in tracer quantities are used. b. Therefore, this request seeks approval only for use of the specified radioisotopes, the experiments otherwise not requiring individual approval under AR 70-25. However, sufficient description is furnished to indicate importance of the studies in warranting use of radioisotopes. c. For any studies later contemplated under the general description given in this request which would, in and of themselves (apart from the use of radioisotope tracers), constitute unusual or hazardous experiments, specific approval (directed to the non-isotope aspects) would then be requested per AR 70-25. d. This request will present the health physics aspects of the radioisotope tracers required (Section II); will present in detail the research methods and plans for C-14 and H-3 usage in vitamin C 2 studies, and outline other proposed studies referring to the vitamin C studies as a model (Section III, Par. 11); and will describe other metabolic and nutritional studies requiring other radioisotope tracers in addition to C-14 and H-3 (Section III, Pars. 12-15). 3. General guidelines for requested studies a. The administered radioactive material would in no case exceed a radiation dose high enough to approach the permissible dose indicated in CFR Title 10, Part 20, RC-12, "The Medical Use of Radioisotopes--Recommendations and Requirements by the Atomic Energy Commission." In fact, in no case will the dose exceed one-half that of the permissible dose and every attempt will be made to use even lesser amounts of isotope when compatible with obtaining reliable data. b. All policies, procedures and regulations prescribed in AR 70-25 and AR-40-37 will be rigidly adhered to in all investigations. c. The person in charge of each specific phase of the studies proposed herein will be formally designated prior to the research by the Commanding Officer, USAMRNL, from the Government scientists listed in this application, and the attending physician will similarly be designed from the Medical Officers among them. 4. History of USAMRNL isotope usage a. This laboratory has employed radioactive labeled compounds in studies with human subjects under AEC License Number 5-46-6 since 17 December 1957. Authorization was given initially to use Iodine131 labeled 3 human serum albumin to measure the turnover rate of albumin of 10 normal young men in various nutritional states. b. USAMRNL staff members have had experience in the use of various radioisotopes in a number of chemical forms in collaborative clinical investigations with Fitzsimons General Hospital involving the basic disease process or new treatment procedures. Such work has been carried out under the Fitzsimons General Hospital's AEC License 5-46-9 which includes authorization for use of the following: (1) Iodine131 for diagnosis of thyroid function and thyroid scanning. (2) Iodine131 labeled human serum for the determination of blood volumes and plasma volumes. (3) Iodine131 labeled Rose Bengal dye for determination of liver function and liver scans. (4) Iodine131 labeled fats and fatty acids for determination of fat absorption. (5) Iodine131 labeled renal function compounds. (6) Phosphorus32 for the treatment of polycythemia vera, leukemia and bone metastasis. (7) Chromium51 for the determination of red cell volume and red cell survival time. (8) Cobalt60 labeled vitamin B12 for the diagnosis of pernicious anemia. 4 (9) Iron59 for iron metabolism studies. c. On 11 December 1959, authorization was granted in License No. 5-46-12(L 61) for the use of Carbon-14 labeled glucose, glucuronic acid, glucuronolactone and ascorbic acid to measure the pool size and turnover rate of body ascorbic acid in normal human subjects and for investigation of the possibility that humans may be able to synthesize small amounts of ascorbic acid. d. License No. 5-46-12(L 61) was renewed on 24 October 1961 and expanded to include Carbon-16 labeled glycine, cholesterol, mevalonic acid acetate and carbon monoxide, in addition to the compounds previously authorized, for use in metabolism and physiological tracer studies in humans. e. A recent amendment to License No. 5-46-12 (including prior approval by the Secretary of the Army) permitted the use of tritiated water for the determination of total body water in 112 human volunteers at Ft. Carson, Colorado. f. Current status of AEC radioisotope licenses of USAMRNL and Fitzsimons General Hospital: (1) This laboratory is current licensed by AEC (License No. 5-46-12 (L 63) for human use of the isotopes listed above in Par. 4c and d; the specifically authorized study in Par. 4e having been completed. All human usage not explicitly covered by Par. 4c of AR 70-25 has been discontinued pending authorizations (requested by this document) required by AR 40-37. This AEC license will expire 31 December 1963. 5 (2) This laboratory also currently operates under the general (animal usage) AEC radioisotope License No. 5-46-11 (H 63). This was originally scheduled to expire 31 August 1963, but has been extended indefinitely by AEC (who are holding our renewal application dated 21 May 1963) pending consolidation of the separate FGH and USAMRNL licenses into one broad license for the entire post (a joint FGH and USAMRNL license). (3) Consolidation of the heretofore separate licenses of FGH and USAMRNL into one broad license has been recommended by the Preventive Medicine Division of Office of The Surgeon General, and sanctioned by AEC because of the favorable record of radioisotope handling by both FGH and USAMRNL. Application for the new joint license (omitting the radioisotopes requested herein for volunteer research use) will be forwarded to AEC (through The Surgeon General) by FGH-USAMRNL within approximately 10 days. (4) Upon approval of the radioisotopes requested herein per Par. 3b(3) AR 40-37, application will be made to AEC (through The Surgeon General) for addition to the joint AEC license by amendment. 5. Specific radioisotopes to be used a. Use of the following radioisotopes in volunteer human research in tracer dosages is requested: By-product Material Chemical and/or Physical Form Carson-14 Vitamins Amino acids Lipids (as glycerides, cholesterol and free fatty acids) Carbohydrates Acetate Mevalonic acid Bicarbonate or CO2 6 By-product Material Chemical and/or Physical Form Hydrogen-3 Vitamins Magnesium-28 MgO,MgC12, Mg citrate Calcium-47 CaCl2 Calcium-45 CaCl2 b. All the labeled compounds to be employed are naturally occurring nutrients or metabolites for the human. 7 Section II. General Health Physics for Requested Isotopes 6. Carbon-14 Carbon-14 has a soft beta emission that lends itself to tracer studies. Fat in the body is usually considered the critical organ. The biological half life for Carbon-14 in fat is given as 35 days. The National Bureau of Standards Handbook No. 69 lists the maximum permissible burden in fat as 300 uc. Constants for calculating maximum permissible internal concentration of radioisotopes assumes that 50% of the Carbon-14 that is present in the blood is transferred to the critical organ, fat. However, based on animals, it can also be assumed that few of the Carbon-14 labeled compounds proposed to be used would approach this retention in the critical organ. The majority of the compounds proposed are readily metabolized and removed from the body as expired CO2 or metabolites in the urine, and would reduce even further the body burden of irradiation. Flushing procedures could also be employed in the case of the labeled vitamins to hasten their removal from the body upon completion of the studies. In all investigations, balances will be performed that will permit careful knowledge of the extent of retention and turnover of the labeled compound administered. 7. Hydrogen-3 Hydrogen-3 emits only a very soft beta particle, but with present counting instruments is a very useful isotope for tracer studies. The entire body is generally considered the critical organ and the isotope has a biological half life of approximately 12 days. The maximum permissible body burden is 1-2 mo. This approximate 8 amount has been used routinely in numerous laboratories for the determination of total body water in the human. Permission has been granted this laboratory to use this technique utilizing one millicurie of tritiated water on volunteers at Ft. Carson. The use of tritiated vitamins is proposed since several vitamins are available only as the tritiated compounds. Because of the considerably smaller pool size, the dosage of tritium employed as a vitamin will be must less than that employed in the measurement of total body water. Amounts less than 0.1 mc are anticipated. Tritiated folic acid and pyridoxine are presently employed at a number of laboratories for studying malabsorption syndromes in humans such as may be encountered in tropical sprue. 8. Magnesium-28 This isotope is available as a cyclotron produced element. It has a very short half life of only 21 hours. Magnesium-28 has been used in a number of laboratories with humans. Dr. J. K. Aikawa, Department of Medicine, University of Colorado School of Medicine, Denver, has administered 90 us of Mg-28 to normal subjects and patients and found essentially no activity in the urine or plasma after 40 hours. By this time, approximately 90% of the Mg-28 was accounted for in the feces and urine. (Peaceful Uses of Atomic Energy, Vol. 24, p. 148, 1958; The Role of Magnesium in Biological Process, J.K. Aikawa, 1963, C.C. Thomas, Publishers, Springfield, Ill.) 9 9. Calcium-47 This relatively recent available isotoped with a half life of only 4.9 days has seen use in a number of studies with human subjects. The maximum permissible burden when the total body is considered the critical organ is approximately 10 uc; with bone the critical organ a permissible burden of 5 uc is allowed. For the proposed studies, a dose not to exceed 5 uc would be used, with an anticipation that a dose of only 2 uc may be sufficient. 10. Calcium-45 If the use of Calcium-47 should prove not feasible because of the short half life and transportation or delivery difficulties, Calcium-45 would be employed instead. Calcium-45, with a soft beta emission and a half life of 163 days, has a maximum permissible burden in bone of 30 uc or 200 uc for the total body. The dosage proposed for the studies outlined would not exceed 15 uc. All use of radioisotopes in humans would be in accordance with the following: 1. Use will be confined to metabolic and physiological tracer studies. 2. The licensee shall comply with the provisions of Title 10, Part 20, Code of Federal Regulations, Chapter 2, "Standards for Protection Against Radiation," and RC-12 "The Medical Use of Radioisotopes--Recommendations and Requirements by the Atomic Energy Commission." 10 3. Radioisotopes for use in humans shall be acquired from a supplier other than an Atomic Energy Commission facility, who certifies the pharmaceutical quality and assay of such material. 4. The licensee, except as otherwise specifically provided for in the license, shall possess and use the material as prescribed in this license in accordance with statements, representations and procedures contained in supplementary sheets attached to the application. 5. All rules, regulations and limitations set forth by Army, AEC, and local authorities (including those set forth in AR 70-25, AR 40-37 and Handbook 69 of the National Bureau of Standards) will be complied with. 11 Section III. Proposed Nutrition and Metabolism Tracer Studies 11. Vitamins: Investigations on the vitamin requirement of the human with the use of Carbon-14 or Hydrogen-3 labeled vitamins or related compounds (References cited in the paragraph (11) are listed in subparagraph 11(h) a. Background for vitamin C studies Past studies (1) indicated that D-glucuronolactone caused increased blood ascorbic acid levels as well as increased urinary excretion of ascorbic acid in men, whereas D-glucuronic acid did not do this. To check the possible conversion of D-glucuronolactone to ascorbic acid, it was decided to study the metabolism of the lactone in two ways. One was to give the D-glucuronolactone-6-C14 orally and then isolate urinary ascorbic acid to determine if any of the labeled lactone had been converted to L-ascorbic acid. The other was first to label the total body ascorbic acid pool with L-ascorbic-1-C14 acid and then test with various loads of D-glucuronolactone to see if any changes would take place in the specific activity and rate of excretion of ascorbic acid. Further, an attempt was made to see if total body ascorbic acid and its rate of utilization were related to the fat-free body weight. Results of studies (2) with healthy men revealed that close to one-fourth of D-glucuronolactone-6-C14 was converted to L-ascorbic acid whereas, on the other hand, no activity could be detected in the ascorbate derivative isolated from the urine of subjects receiving D-glucuronic-6-C14 acid. In addition, it was found that one-half of the urinary oxalate arises from the breakdown of ascorbic acid and is excreted at a constant rate. Further in 6 men of 12 diverse body weight and degree of fatness, it was found that ascorbate utilization, as expressed in terms of C14 oxalate excretion, occurred at a rate of 0.207 mg per day per kilogram of fat-free body weight. One of the more interesting findings of these experiments was that no C14O2 activity could be detected in the expired air of the subjects receiving L-ascorbic-1-C14 acid even with the use of a 15-liter ionization chamber for greater sensitivity. A fraction of 1% oxidation to CO2 during the first 8 hours could have been easily detected by this technique. This finding was in agreement with the earlier work of Hellman and Burns (3). Recently, Abt et al. (4) reported that a man excretes approximately 25% of the total activity of L-ascorbic-1-C14 acid via the lung. Because doubt had been caused as to whether or not man could decarboxylate C14 labeled ascorbic acid to C14O2, a series of experiments were performed in this laboratory which resulted in a publication (5) showing: 1. Chromatographic and radioautographic evidence was presented showing that progressive degradative changes occur in L-ascorbic acid dissolved in water and kept at 25ø C. for a 72-hour period; 2. When a human subject received 20 uc of freshly dissolved L-ascorbic-1-C14 acid solution, little or no C14 appears in his respiratory CO2; 3. Men who were given similar samples of L-ascorbic-1-C14 acid aged for 36 and 72 hours, respectively, excreted 30.6% of the ingested C14 as respiratory CO2. The true nature of the compounds undergoing decarboxylation in man in these studies cannot be defined from the work presented here except that they are not reduced ascorbic acid. 13 b. Request for use of Carbon-14 to label vitamin C and related compounds. Therefore, because of demonstrated usefulness and necessity of using tracer techniques to study metabolic pathways, the proposed is being made that tracer amounts of Carbon-14, as glucose-6-C14, glucuronolactone-6-C14, glucuronic-6-C14 acid and ascorbic-1-C14 acid be administered by mouth to humans in further studies for the purpose of measuring the pool size and the rate of utilization of body ascorbic acid under varying conditions. The subjects to be used will be military personnel (volunteering for the specific study) or laboratory personnel, both male and female, or Fitzsimons General Hospital personnel (as well as Fitzsimons General Hospital patients who volunteer). The possible hazards of the experiments will be explained in advance to all subjects. Although multiple experiments may be performed on individuals, in no case will the total body radiation dose from this experiment, other experiments, or from x-rays, exceed the maximum possible limits for normals of 5 rem per year (lower below age 25). c. Experimental methods (using labeled vitamin C and related compounds The L-ascorbic-1-C14 acid will be obtained from the California Corporation for Biochemical Research, Los Angeles. All C14 labeled compounds will be checked for purity prior to use by melting point measurement and by paper chromatography. The activity of all C14 labeled compounds will be checked by radioassay. The 14 L-ascorbic-1-c14 acid will be freshly dissolved in distilled water and immediately swallowed by the experimental subject. No cold carrier will be given to these subjects. Total daily urine will be collected and measured from all subjects; these samples will be refrigerated and 2.0 ml of each will be taken for radioassay. Immediately after receiving the tracer quantity of L-ascorbic acid, the subjects will be made to expire directly through a CaCl2 drying train into a 5-liter Cary-Tolbert ionization chamber connected to a vibrating reed electrometer. The C14O2 activity, total CO2, and the volume of flow is recorded automatically on a 6-channel recorder. The total activity of each urine sample is determined by use of a liquid scintillation counter using P-dioxane-toluene. Oxalate in selected samples is isolated as calcium oxalate, recrystallized 4 times and dissolved in 1 N hydrochloric acid for counting in the liquid scintillation counter. Quantitative determination of the total oxalate is done by the Archer method(6). Efficiencies for all liquid scintillation counting of samples are determined individually by use of added standard C14 samples. Urinary ascorbic acid levels are chemically determined by the Schaffert method (7). Urinary ascorbic acid is the - isolated by the method described by Jackel et al.(8). After the dinitrop-enylhydrazone (DNPH) derivatives are recrystallized, they are dissolved - P-dioxane and applied to weighed planchets and counted in a gas flow counter. All DNPH derivatives are recrystallized to constant activity which usually requires 4 to 6 recrystallizations. 15 Total body tissue volume (V) is estimated in duplicate tests using a body volumeter based on displacement of water (9). From body weight (M) and V, fat (F) in kg is calculated according to an equation developed in this laboratory: F = 4.834 V - V.336M. d. Experimental plan, studies on factors that may influence the vitamin C metabolism and requirements in man: (1) Recapitulation of the method Studies of body composition and the use of C14 isotopes have resulted in a method for stating the actual utilization of ascorbic acid by healthy men. In human subjects who ingest 20 uc of L-ascorbic-1-C14 acid, the daily urinary oxalate arising from metabolism of the labeled ascorbate is subsequently excreted as a constant proportion of total C14 activity remaining in the body. Thus, it can be inferred that the portion of the daily oxalate which arises from metabolism of ascorbate is formed and excreted at a constant rate. Ingestion of a single, comparatively large 0.5 gm quantity of unlabeled ascorbic acid or its precursors by subjects whose body ascorbic acid pools had been previously labeled, as described above, results in increased excretion of C14 ascorbate of lowered specific activity. These effects are transitory in that within 2 days total ascorbate excretion returns to previous levels and ascorbate specific activity is lower than it was prior to dilution of the body ascorbate pool. 16 Simultaneously, the total activity and the specific activity of the oxalate decrease, but the proportionality of total oxalate activity to specific activity of the ascorbate remains the same. From these effects, it can be inferred that the utilization of breakdown of ascorbic acid in the body occurs at a constant rate irrespective of an increased rate of supply of ascorbate to the body. Further, in 8 men of diverse body weight and degree of fatness, it was found that ascorbate utilization, as expressed in terms of C14 oxalate excretion, occurred at a rate of 0.207 mg per day per kg of fat-free body weight. Rarely, if ever, do adult males exceed 90 kg in lean body mass. Therefore, 28 mg per day intake would match the greatest quantity of ascorbate metabolized by the largest healthy man. Further, it is of interest to note that despite repeated reports in the literature of loss of ascorbic acid in sweat, when one of the subjects discussed above was seated for a 6-hour period in a hot room after being labeled with 20 uc of ascorbic-1-C14 acid, no E14 activity could be detected in the collected total body sweat. The chemical analysis of the sweat indicated the presence of a small amount of ascorbate. However, when the sweat was lyophilized to dryness and then applied to a chromatographic sheet and run in a standard solvent system, no reduced ascorbic acid could be demonstrated. These results are not surprising in view of the fact that it is well known that all the chemical determinations for ascorbic acid are not absolutely specific for ascorbic acid in biological fluids. 17 The method as employed consists of giving an individual single oral dose of 20-50 uc of L-ascorbic-1-C14 acid and then collecting a single 24-hour urine sample. The ascorbic acid contained in the urine is isolated as the dinitrophenylhydrazine derivative and counted to obtain specific activity as uc/mg of ascorbate excreted. The oxalate that is derived from the labeled ascorbate and excreted is also isolated and counted. Then, by simply dividing the specific activity of the excreted ascorbate by the total C14 activity of the formed and excreted oxalate, one can obtain an estimate of the number of milligrams of ascorbate utilized during the 24-hour period. This method could be used in human studies to determine whether or not there is an increased utilization or an increased need for ascorbic acid in the following conditions: Cold Heat Acclimatization to heat, cold, stress and altitude Stress Trauma and burn patients Infections Moreover, this method could be used in human studies to determine whether or not adaptation occurs in people who have been on a chronic low dietary intake of vitamin C. 2. Need for more data on vitamin C metabolism According to the ext "World Review of Nutrition and Dietetics" (Vol. III, 1963) published by G. H. Bourne, p. 187, the following conclusions regarding vitamin are stated: 18 "1. The most frequently quoted recommended allowance of vitamin C for adult man under the prevailing conditions of civilization and climate varies around 80 mg L-ascorbic acid/day. "2. High doses, though not toxic are not recommended and may, according to some findings, lead even to a negative adaption of the organism. "3. Medium doses (up to 200 mg) are probably reasonable under some special conditions-certain types of work, rehabilitation and therapeutic allowances." At a recent meeting held by the Federation of American Societies for Experimental Biology in Washington, D.C. on 14-15 March 1963, the Ad Hoc Committee on Military Applicability of Research on Ascorbic Acid made the following recommendation: That this laboratory, i.e. USAMRNL, attempt to study the utilization of vitamin C in humans in the following conditions: Cold Heat Acclimatization Stress Interrelationship of vitamin C with other vitamins Wounds and burns (3) Extension of the methods to problems stated in preceding paragraph In view of the above recommendations and the lack of information on the above problems, it is requested that vitamin C utilization studies in the above-named conditions, using the method previously described, in human volunteer subjects be considered for authorization. To accomplish this request, it would require that a team of 2 or more investigators from this laboratory be sent to several geographical areas with differing climatic conditions. The areas under consideration are (1) Camp Hale, Leadville, 19 Climax or Mt. Evans areas in Colorado for studies on ascorbic acid in acclimatization to cold, and (3) a tropical area within the Caribbean Command for studies on acclimatization to tropical conditions. In addition to this general approval for use of isotopes, the approval and concurrence of the local commander or appropriate local health authorities would be obtained for each location and experiment. At each location, comparisons would be made between subjects who had recently arrived and those who had resided at the location for an extended period of time. These results would in turn be compared with findings obtained at this laboratory on subjects residing in Denver, Colorado. At each location, not more than 10 subjects would be studied. Normal, healthy volunteers, preferably military, would be selected to receive the C14 labeled ascorbic acid as previously outlined; body composition data would be obtained by skinfold or other appropriate measurements. With the data obtained from these studies, one could then assign what would be the ideal vitamin C pool size and utilization under these climatic conditions in comparison with the data previously obtained at this laboratory on normal healthy subjects. Upon evaluation of the data, considerations would be made as to recommended allowances for vitamin C under conditions of cold, heat, altitude and acclimatization. Studies on the interrelationship of vitamin C with other vitamins would be performed at this laboratory with normal 20 volunteers, employing C14 labeled ascorbic acid in the amounts and manner as previously outlined. Evidence of a relationship between vitamin C and vitamin B6 in the human has been recently obtained at this laboratory in non-isotopic studies. The use of C14 ascorbic acid in these investigations would permit a better understanding of the apparent interrelationship. The possible increased needs for vitamin C in the situations of stress (surgery or radiation therapy, as examples), wounds or burns would be performed in conjunction with Fitzsimons General Hospital should suitable patients become available. e. Proposal to use C-14 and Hydrogen-3 labeled vitamins other than vitamin C, using the outlined vitamin C studies as a general model Other Carbon-14 labeled vitamins would be studies in essentially the same manner and employing the same techniques and procedures as those indicated for ascorbic acid. Excretion rates, pool size, turnover rates, absorption and metabolic products will be measured for each. The influence of various nutritional states on the above parameters will be investigated in an attempt to evaluate dietary requirements for vitamins. The body pool size of ascorbic acid is considered greater than that of any other vitamin and the turnover rate is as slow or slower than other vitamins; therefore, the amount of radioactive label used for the other vitamins will be less, and with the greater turnover rate, will produce less of a body burden that the vitamin C. 21 When Carbon-14 labeled vitamins are not available, the above studies will be performed with the use of tritium labeled vitamins. The most commonly employed tritiated vitamins are pyridoxine and folacin. These two vitamins have been employed at various laboratories in malabsorption studies with humans. The same parameters and procedures as outlined for Carbon-14 labeled vitamin C and the above will be employed, except that electrometer measurements of expired air will be omitted. The dosage employed will in no case exceed 100 uc of tritium labeled vitamin. This dosage of tritium represents only10% of that routinely employed in total body water measurements and is indicative of the law radiation dose received. f. Health physics The dose of any of the C14 labeled vitamins will not exceed 50 microcuries. The following maximal radiation dosages are calculated with the aid of the ICRP Handbook (Appendix I, reference 1.) The physical half time of C14 is considered infinite. The following is an approximation of the biologic half time. All the vitamins to be used are highly reactive; however, for ascorbic acid the turnover rate is probably slower, possibly much slower than for the other vitamins. From nutritional data, the total body ascorbic acid is almost certainly less than 6 gm, and the daily turnover greater than 10 mg. The half time of body vitamin C under these conditions would be 400 days. This 22 estimate is obviously too long, perhaps by as much as an order of magnitude. The only data in the literature on man (L. Hellman and J. J. Burns, J. Biol. Chem. 230: 923, 1958) (E.M. Baker, H.E. Sauberlich, S. J. Wolfskill, W. T. Wallace and E. E. Dean, Proc. Soc. Exp. Biol. and Med. 109: 737, 1962) shows a pool size of about 1.4 gm in a 70 kg man, with a half time of about 16 days. With the maximum estimate of a 400-day half time, a dose of 50 microcuries of C14 ascorbic acid evenly distributed in a 70-kg man will give a total radiation dose of only 1.13 rem (0.164 rem the first 13 weeks). With a half time of 16 days, the same dose will give a total radiation dose of 0.045 rem (0.044 rem the first 13 weeks). In the case of tritiated vitamins, the dosage will in no case exceed 100 uc of tritium. This dosage of tritium represents only 10% of that routinely employed in total body water measurements; consequently, the body burden is very low. g. Personnel For each specific phase of the studies, one of the following (other than the consultant) will be designated as project leader, and one of the Medical Officers named below will be designated as attending physician per Par. 6, AF 70-25. Maj. E. M. Baker, Ph.D., MSC H.E. Sauberlich, Ph.D. (PL-313) Lt. Col. M. E. McDowell, M.D., MC Lt. Col, J. E. Hansen, M.D., MC Capt. J. R. Handy, M.D., MC B. M. Tolbert, Ph.D. (Consultant, University of Colorado) 23 h. References (1) Baker, E. M., E. L. Bierman, I. C. Plough. Metabolism 9: 478, 1960 (reprint attached, Appendix IV). (2) Baker, E. M., H. E. Sauberlich, S. J. Wolfskill, W. T. Wallace and E. E. Dean, Proc. Soc. Exp. Biol. and Med. 109: 737, 1962 (reprint attached, Appendix IV). (3) Hellman, L. and J. J. Burns, J. Biol. Chem. 230: 923, 1958. (4) Abt, A. F., S. Von Schuching and L. Enns, Am. J. Clin. Nutrition 12: 21, 1963. (5) Baker, E. M., N. G. Levandoski and H. E. Sauberlich. Proc. Soc. Exp. Biol. and Med. 113: 379, 1963 (reprint attached, Appendix IV). (6) Archer, H. E., AA. E. Dormer, E. F. Scowen and R. W. E. Watts. Clin. Sci. 16: 406, 1957. (7) Schaffert, R. R. and G. R. Kingsley, J. Biol. Chem. 212: 59, 1955. (8) Jackel, S. S., E. H. Mosbach and C. G. King. Arch. Biochem and Biophys. 31: 442, 1951. (9) Allen, T. H., H. J. Krzywicki, W. S. Worth and R. M. Nims, U. S. Army Med. Rsch. Nutrition Lab. Rpt. No. 250, 24 Sept. 1960. (10) A study of the military applicability of research on ascorbic acid. Life Sciences Research Office, Federation of American Societies for Experimental Biology, Washington, D.C., August 1963. 24 12. Amino Acids: Investigations on the metabolism of amino acids in the human with the use of Carbon-14 labeled compounds (references cited in this paragraph are listed in Par. 12F) a. Background The recent work of Crawhall et al. (1) using 1-C13 - glycine has demonstrated that this isotope was diluted about 2 1/2 times during the conversion of glycine from the first metabolic pool (i.e. the pool of glycine with which a dose of glycine mixes immediately after absorption and distribution, and which can be sampled by means of the uncombined urinary glycine (2) to oxalate, indicating that about 40$ of the urinary oxalate was derived from glycine during this period. Berlin et al. (3) did not measure the C14 activity in the urinary oxalate in their glycine-2-C14 studies. However, they did show with the use of the methyl labeled glycine that 90% of the C14 activity was accounted for in the expired C14O2 and that only 5% of the C14 activity was excreted in the urine. This would tend to indicate that the catabolism of glycine-2-c14, insofar as oxalate formation is concerned, is far different than that of the carboxyl labeled glycine-1-C14. When C14 labeled ascorbic acid was orally administered to humans, Hillman and Burns (4) reported that an 25 average of 44% of the total radiocarbon excreted in urine was recovered as oxalate. It was demonstrated in this laboratory that 50% of the urinary oxalate was derived from L-ascorbic acid-1-C14 and was excreted at a constant rate per day (5, 6). Therefore, it is of interest to study both glycine-1-C14 and glycine-2-C14 metabolism in humans to determine (1) whether or not the glycine C14 is partially converted to and excreted as oxalate at a constant rate per day as well as (2) the amount per day excreted as urinary oxalate. Further, it would be desirable to measure the expired C14O2 in a vibrating reed electrometer to determine the amount and extent of decarboxylation of the C14 labeled glycine in man. It should be noted that this has been done in humans using glycine-2-C14 (3), but not with the glycine-1-C14. b. Experimental plan (Part 1) A total of no more than 10 human subjects would be involved in these experiments. Further, the subjects would be staff members of this laboratory, 20-46 year-old males. There would be no dietary restriction placed on these subjects. Each subject would have to receive orally 20 uc of glycine-1-C14 as well as a further 20 uc of glycine-2-C14 at a much later date (40-80 day interval). No cold carrier glycine will be given to the subject at the time the labeled material is administered. Immediately after taking the C14 glycine, the subject will be made to breathe through a drying train directly 26 into the ionization chamber of the vibrating reed electrometer. The subject will continue to breathe at intervals through the system until he reaches his background trace signal. This is done by having the subject breathe into the system for 20-30 minutes, then allowing a 30-minute rest. This process is continued until the electrometer tracing returns to background signal. Further, 24-hour urine collections will commence with the ingestion of the C14 glycine label and will continue on every other 3rd day for a period of 2 weeks. The urine samples will then be analyzed for the total urinary oxalate content. The oxalate of each sample will then be isolated and counted in the liquid scintillator. Also, certain selected samples of the urine will be analyzed on the amino acid analyzer which has a flow-through scintillation detector attached. This will enable us to obtain both the specific and total activity of the urinary free glycine of other radioactive metabolites. Each urine sample will be counted for total activity. Thus, if one knows the total dose given as glycine C14 as well as the dose remaining in the body at any given time, as well as the specific activity of the excreted urinary glycine, one should be able to approximate the total body pool size and turnover rate of the free glycine pool. c. Health physics pertaining to Par. 12b The normal A.E.C. procedures shall be adhered to insofar as the administration, handling of the isotope and the 27 disposal of the urine samples obtained from the subjects. The dose of the C14 glycine will not exceed 20 uc for either the glycine-1-C14 or the glycine-2-C14. The following maximal radiation dosages are calculated with the aid of the ICRP Handbook (Appendix I, reference 1). The physical half life of C14 is considered infinite. The following is an approximation of the biologic half time of glycine-2-C14. According to N. I. Berlin, B. M. Tolbert and C. Lotz (J. Clin. Investigation 31, No. 3: 335-337, 1952), the longest "half time of glycine-2-C14 elimination from the tissues of man is approximately 50 days." With the estimate of a 50-day half time, a dose of 20 uc of glycine-2-C14 evenly distributed in a 70 kg man will give a total radiation dose of only 0.056 rem (0.0478 rem the first 13 weeks). Another body compartment of considerably less importance and a half time of about 100 days was described at a later date by Berlin et. al. (Proc. Soc. Exp. Biol. and Med. 88: 386, 1955). However, since the majority of the dose is not retained, but lost within the first 24-hour period as expired CO2 as urinary excretory products, the body irradiation burden is considerably less than this value. In the case of glycine-1-C14, we have only the date of R. W. E. Watts and J. C. Crawhall (Biochem. J. 73: 277-86, 1959) using the stable C13 isotope to estimate the glycine metabolic pool in man. According to these authors, the pool size of glycine in a 70 kg man is 406 gm or 5.8 gm/kg. Further, they state that the turnover rate in a 70 kg man is 3.2 gm/hr. or 76.8 gm/day. Thus, 76.8 = 0.189 or 18.9% turnover. The ---- 406 biological ö 1/2 would then be equal to 0.693 = 3.7 days. Assuming ----- 0.0189 then that 4-day life for the glycine-1-C14, a dose of 20 uc evenly distributed in a 70 kg man will give a total radiation dose of only 0.005 rem (0.005 rem in the first 13 weeks). 28 d. Experimental plan Part II) Once Carbon 14-labeled amino acids would be studies in essentially the same manner as that employed with glycine. Similarly, pool size, turnover rates and metabolites would be measured in an attempt to study the protein and amino acid requirements of the human. These studies should also provide additional knowledge as to the metabolic pathways and interrelationships of amino acids in the human. It is anticipated that not more than 3 subjects will be required for each amino acid investigated. The dosage of Carbon-14 employed would not exceed that indicated for Carbon-14 glycine, and with the half time estimated not to exceed that for glycine. The radiation burden, therefore, would be low and would not in any instance approach the maximum permissible dose. e. Personnel Maj. E. M. Baker, Ph.D., MSC, Project Leader H.E. Sauberlich, Ph.D. (PL-313) Co-Project Leader G. A. Leveille, Ph.D. Lt. Col. M. E. McDowell, M.D., MC (Serving also as attending physician) Lt. Col. J. E. Canham, M.D., MC (Serving also as attending physician) f. References (1) Crawhall, J.C., R. R. Mowbray, E. F. Scowen and R. W. E. Watts, Conversion of glycine to oxalate in a normal subject. Lancet, Nov. 14, 810-11, 1959. (2) Watts, R. W. E. and J. C. Crawhall. The first glycine metabolic pool in man. Biochem. J. 73: 277-86, 1959. (3) Berlin, N. I., B. M. Tolbert and J. H. Lawrence. Studies in glycine-2-C14 metabolism in man. I. The pulmonary excretion of C14O2. J. Clin. Investigation 30: 73-76, 1951. 29 (4) Hillman, L., J. J. Burns. Metabolism of L-ascorbic acid-1-C14 in man. J. Biol. Chem. 230: 923-930, 1958. (5) Baker, E. M., H. E. Sauberlich and S. J. Wolfskill. Metabolism of D-glucuronolactone-6-C14 and D-glucuronic acid-6-C14 in man. Fed. Proc. 20: 85, 1961. (6) Baker, E. M., H. E. SAUBERLICH and S. J. Wolfskill, W. T. Wallace and E. E. Dean. Proc. Soc. Exp. Biol. and Med. 109: 737, 1962. 30 13. Lipids: Studies on lipid metabolism in the human with the use of Carbon-14 labeled compounds (references cited in this paragraph are listed in Par. 12c) a. Experiment 1: Carbon-14 tracer studies on cholesterolmetabolism in the human (1) Background and procedures Tracer amounts of C14, as cholesterol-4-C14, will be administered orally to study the influence of neomycin and various fats on cholesterol absorption in order to determine whether the hypocholesteremic effect of these materials is the result of an impaired absorption. In order to ascertain whether these materials influence cholesterol synthesis, acetate-1-2-C14 and mevalonic acid-2-C14 will be administered intravenously. The work of Samuel and Steiner (Proc. Soc. Exp. Biol. Med. 100: 193, 1959) has demonstrated a hypocholesteremic effect for neomycin. Unsaturated fat has also been shown to have a cholesterol lowering effect. The mechanisms by which neomycin or unsaturated fat depress plasma cholesterol remain obscure. Unpublished data from this laboratory indicate the neomycin functions by interfering with cholesterol and/or bile acid absorption. The effect of unsaturated fats appears to be twofold: a) an interference with cholesterol absorption mediated by the sterol fraction in vegetable fats and b) a particular systemic effect (Bronte-Stewart, Fed. Proc. 20: No. 1, Part III, p. 127, 1961). In order to further elucidate the mechanism of action of these compounds, human volunteers fed neomycin, different fats or a control diet will be given an oral dose of cholesterol-4-C14 31 (20-50 uc) and its absorption determined. In other subjects, similarly treated, acetate-1-2-C14 or mevalonic acid-2-C14 will be administered intravenously (50 uc) and incorporation into cholesterol will be ascertained by determining the specific activity of plasma cholesterol. (2) Health physics The maximum dosage to be employed is 50 uc for acetate, mevalonic acid and cholesterol. The maximal radiation dosage, calculated with the aid of the ICRP Handbook (Appendix I, reference 1) for these levels of administered radioactivity, will not exceed the permissible limits for normal subjects of 5 rem/yr. with n o more than 3 rem in any 13 consecutive week period (above age 18). The half life of cholesterol is approximately 20 days (Cook, R. P.: Cholesterol, 1958, Academic Press, N. Y.) and if that of other compounds synthesized from acetate or mevalonic acid is assumed to be similar and, further, the physical half life of C14 is considered infinite, a dose of 50 uc of C14 labeled cholesterol, mevalonic acid or acetate evenly distributed in a 70 kg individual will give a total radiation dose of 0.056 rem (0.054 rem the first 13 weeks). (3) Personnel Gilbert A. Leveille, Ph.D., Project Leader Howerde E. Sauberlich, Ph.D., Project Leader Lt. Col. M. E. McDowell, M.D., MC (Serving also as attending physician) Lt. Col. J. E. Canham, M.D., MC (Serving also as attending physician) 32 b. Experiment 2: Suppressibility of cholesterol synthesis by exogenous cholesterol loading in man (1) Background The relative stability of serum cholesterol levels, despite marked variation in dietary intake of cholesterol, has been attributed to compensatory changes in hepatic synthesis of cholesterol (1-5). Recently, Sipperstein and Guest have suggested, on the basis of in vitro studies, that the mechanism of this homeostatic effect is a sensitive negative feedback system whereby cholesterol inhibits the conversion of B-hydroxy-B methyl glutaryl Co A to mevalonic acid(6). These authors speculate that insensitivity of this feedback might be involved in disorders of cholesterol metabolism. In order to test this hypothesis, it is planned to ascertain quantitatively the response of cholesterol synthesis to an exogenous cholesterol load. After data on normal subjects have been obtained, these will be compared with groups demonstrating abnormalities of cholesterol metabolism, i.e., idiopathic hypercholesterolemia-proven atherosclerosis, diabetes, hypo-and hyperthyroidosis, nephrotic syndrome. (2) Method Patients will be given 100 microcuries 1-c14 acetate intravenously or orally. Timed serum samples will be analyzed for total and C14 cholesterol. In certain patients, C14 of other 33 serum lipids and C14 as C14O2 will also be measured. These procedures will then be repeated after a standard cholesterol load sufficient to elevate serum cholesterol in normal subjects(7). Differences in total and specific activity of serum cholesterol before and after cholesterol loading will be used as an index of the sensitivity of the hepatic response to exogenous cholesterol. Methods of analysis will be similar to those described by Gould et al. (8). (3) Health physics With regard to regard to radiation safety, reference is made to the work of Gould et al. (8): "The dose of 100 uc was chosen so that repeated doses could be given to the same subject without exceeding accepted values for the maximum permissible dose for man. Our studies of C14O2 in expired air after the administration of 1-C14 acetate demonstrated that approximately 56 per cent of the radiocarbon was eliminated during the first 24 hours.* On this basis, we have made the assumption that a single 100 uc dose will result in the 'retention' of not more than 25 uc of C14 in the slowly exchanging 'fat compartments' of the body. The maximum permissible dose for C14 compounds retained in the body fats is estimated to be 250 uc, according to calculations in Handbook 52 of the National Bureau of Standards.(7) Thus, we believe we are justified in administering, over a period of several months, a maximum of five such doses to human subjects without regard to their life expectancy. "Ref. 7. Maximum Permissible Amounts of Radioisotopes in the Human Body, etc., Nat. Bur. Standards Hand-book, 52, pp. 12 and 18, G. P. O., Washington, D.C., March 20, 1953. 34 "*Shreeve also reported that 56 per cent of the C14 in acetate was eliminated as C14O2 by man at the end of 24 hours. Hellman reported that 60 per cent of the radiocarbon was retained at the end of 24 hours, and 35 per cent at the end of the first week after administration of acetate. It should be noted that he used the methyl-labeled acetate (2-C14-acetate)." In the present study, only 2 doses of 100 microcuries each will be given instead of 5 doses of 100 microcuries as above. The dose will therefore be well below the maximal permissible dose quoted. 4. Personnel Gilbert A. Leveille, Ph.D., Project Leader Howerde E. Sauberlich, Ph.D., Project Leader Lt. Col. M. E. McDowell, M.D., MC (Serving also as attending physician) Lt. Col. J. E. Canham, M.D., MC (Serving also as attending physician) c. References (1) Gould, R. G. and C. B. Taylor, Fed. Proc. 9: 179, 1950. (2) Taylor, C.B. and R. G. Gould, Circulation 2: 467, 1950. (3) Frantz, I. D., H. S. Schneider and B. T. Henkelman, J. Biol. Chem. 206: 465, 1954. (4) Tomkins, C. B., N. Sheppard and I. L. Chaikoff, J. Biol. Chem. 201: 137, 1953. (5) Hotta, S. and I. L. Chaikoff, Arch. Biochem. 56: 28, 1955. (6) Sipperstein, M. and M. J. Guest, J. Clin. Investigation 39: 643, 1960. 35 (7) Connor, W. E., R. E. Hodges and R. E. Beiber, J. Clin. Investigation 40: 894, 1961. (8) Gould, G. R., G. V. LeRoy, G. T. Okita, J. J. Kabara, P. Keegan and D. M. Bergenstat, J. Lab. and Clin. Med. 46:372, 1955. (8) Radioisotope Studies of Fatty Acid Metabolism. J. F. Mead and D. R. Howton, 1960, Pergamon Press. 36 14. Carbohydrates: Investigations on the digestibility and metabolism of carbohydrates in the human with the use of Carbon-14 labeled compounds (references cited in this paragraph arelisted in Par. 14e) c. Background Experiments have been in progress for some time at this laboratory investigating the digestibility of Carbon-14 labeled cellulose, hemicellulose and various uncommon sugars in laboratory animals as the rat, hamster and guinea pig (1, 2). Balance studies employing a vibrating reed electrometer to measure the expired carbon dioxide, together with urine and fecal measurements, have demonstrated that the rat may digest as much as 25% of the ingested cellulose (1). In the case of the human, it is considered that cellulose passes through the digestive tract without being attacked by any of the digestive enzymes, though some bacterial decomposition probably takes place in the large intestine. Whether or not the bacterial actions are of value to the human are unclear. Various studies at this laboratory as well as elsewhere would indicate that at times cellulose is digested to a limited extent by the human (3-5). However, it must be recognized that in human balance studies, the methods for the measurement of cellulose and hemicellulose are less than satisfactory for critical evaluation. Furthermore, the possibility of bacterial decomposition of cellulose or hemicellulose in the lower intestinal tract may give rise to an "apparent" digestibility in terms of nutrient benefit to the human. The use of Carbon-14 labeled cellulose with human subjects would give more definitive results with regard to this problem. Very exacting balance studies could be conducted. Furthermore, the presence or lack of presence of radioactivity in 37 the expired carbon dioxide, or in the urine and blood, would be rather conclusive evidence, which could be quantitated, that cellulose is or is not utilized. If utilization is indicated, the question of whether or not it is mediated through the intestinal flora could be readily investigated with the use of oral antibiotics (3, 4). b. Procedures The procedures employed would be very similar to those previously described for use with Carbon-14 labeled vitamin C. Normal, healthy volunteer subjects (minimum number) would receive orally specially prepared Carbon-14 labeled cellulose in an amount not to exceed 100 uc. The subjects will have previously received controlled levels of cellulose in the diet to investigate the influence of this dietary component on the digestibility of the C14 cellulose. Balance studies will be conducted with the aid of markets for the stools. The expired air will be monitored with the aid of a 5 or 15-liter chamber with a Cary-Tolbert vibrating reed electrometer and automatic carbon dioxide measurements. Urine collections will also be made throughout the period and radioactivity measurements performed with a scintillation counter. If significant amounts of radioactivity are found to be present, attempts will be made to determine the nature of the radioactive compounds. If evidence of cellulose digestion is noted, additional subjects will receive prior to receipt of the C14 38 cellulose oral supplements of antibiotics in an effort to study the possible role of the intestinal flora in the digestion process. The antibiotics, neomycin and bacitracin would be employed, using the amounts and procedure previously employed in recent studies by the Metabolic Division of this laboratory to reduce or eliminate the intestinal flora of human subjects (3, 4). If C14 labeled hemicellulose or pectins can be made available, similar digestibility studies would be conducted with these materials. c. Health physics As indicated above, the dosage of the Carbon-14 labeled cellulose, hemicellulose or pectin would not exceed 100 uc. Although the digestibility of these compounds is not fully know, it is exceedingly doubtful that any are completely absorbed. If cellulose is digested to any extent, it would be most likely converted to glucose which the body readily metabolizes with a major portion being removed quickly through the lungs as carbon dioxide. Even if it is assumed that 100% digestion and assimilation takes place, this dosage of radioactivity would be considerably less than the maximum permissible dose. Glucose, the unit component of cellulose, is metabolized in large quantities each day by the human body. The radioactivity would, therefore, be readily diluted throughout the body and not concentrated or localized in a small amount of tissue. The eventual critical organ for what Carbon-14 that 39 would be retained would be fat; but in consideration of the amount of Carbon-14 retained and deposited in the fat and with a biological half life of 35 days for Carbon-14 in fat, the radiation body burden produced by 100 uc of cellulose-C14 would be considerably less than the maximum permissible dose. With the aid of the ICRP Handbook (Appendix I, reference 1), the calculated total dose would be 0.690 rem (0.576 rem the first 13 weeks). Assuming no absorption, the greatest dose received by the intestinal tract would be 0.691 rem. d. Personnel H. E. Sauberlich, Ph.D., Project Leader Lt. Col. M. E. McDowell, M.D., (MC)(Also attending physician Lt. Col. J. E. Canham, M.D., (MC) (Also attending physician) Maj. E. M. Baker, Ph.D. (MSC), Co-Project Leader B. M. Tolbert, Ph.D. (Consultant, University of Colorado) e. References (1) Johnson, R. B., D. A. Peterson and B. M. Tolbert. Cellulose metabolism in the rat. J. Nutrition 72: 353, 1960. (2) Johnson, R. B. Metabolism of cellulose by the normal nonruminant, the rat. U. S. Army Med. Rsch. and Nutrition Lab, Annual Progress Report., p. 254, June 1963, Denver, Colorado. (3) Leveille, G. A., R. C. Powell, H. E. Sauberlich and W. T. Nunes. Effect of orally and parenterally administered neomycin on plasma lipids of human subjects Am. J. Clin. Nutrition 11: 156, 1962. (4) Powell, R. C., W. T. Nunes, R. S. Harding and J. B. Vacca, The influence of nonabsorbable antibiotics on serum lipids and the excretion of neutral sterols and bile acids. Am. J. Clin. Nutrition 11: 156, 1962. 40 (5) Canham, J. E. et al. A study on feeding of a uniform microcrystalline cellulose--its digestibility and effects on digestibility of other macronutrients. U. S. Army Med. Rsch. & Nutrition Lab. Annual Progress Rpt., p. 180, June 1963, Denver, Colorado. 15. Minerals: Studies on mineral metabolism and interactions in the human with the use of radioisotopes (reference cited in the paragraph are listed in Par. 15f) Initial studies would seek the role of magnesium and calcium in human kidney stone disease: a. Background Urinary calculi are among the most ancient afflictions of man. This painful and often fatal disease is known to have occurred as long as 8,000 years ago (1) and no rase or geographical area has been entirely free of a calculus problem. There appear to be "stone belts" of high incidence in regions such as southern China, northern Thailand, the Punjab district of Indian, Arabia and Iraq. In addition, there have been reports of "stone waves," one such occurring in Europe during this century (2). Despite the antiquity and frequency of this disease, the basic mechanisms of calculus formation remain unknown, and fully 85% of all patients who form urinary calculi have no recognized local or systemic disease (3). Most authorities agree that a nutritional deficiency or imbalance is a probable factor, but few reliable studies have been conducted to relate specific nutrients to calculus formation in human populations (4). Studies at this laboratory (5) and elsewhere have established that nephrocalcinosis and urolithiasis (principally phosphates and carbonates of calcium) are frequently associated with magnesium deficiency in rats and other species. Of particular 41 interest is the observation that about 20% of apparently otherwise normal rates consuming a semipurified diet containing 400 ppm magnesium (minimum requirement for growth is 120-150 ppm) will develop uroliths similar to those found in the markedly deficient rat and that elevation of the dietary Mg to 4,000 ppm will present this occurrence. Furthermore, Selye has shown that intraperitoneal administration of magnesium will prevent the formation of uroliths which normally follows experimental hyperparathyroidism in the rat. An increase in dietary magnesium will also markedly lessen the accumulation of calcium in the kidney which results from a high phosphorus intake. Despite these indications on an important role for magnesium in calculus formation and calcium metabolism, practically no published information exists on the metabolism of this nutrient in human urinary calculi disease (4). Preliminary studies at this laboratory (6) have indicated that some populations in areas with a reported high incidence of stone formation (e.g., Burma investigations) may, indeed, consume relatively low amounts of magnesium. In addition, magnesium supplements have brought at least a temporary (6 months) halt to the formation of phosphatic type stones in a patient with no demonstrable infection or metabolic disorder and a previous rate of stone formation of 2 per month for a period of 3 years (7). Based on this evidence, it is felt that considerable justification exists for the study of the role of magnesium as well as other factors in human renal lithiasis. Certainly, a primary 42 objective is a determination of the value of magnesium supplements in a large number of patients and a study of any changes in urinary constituents associated with a favorable response to this therapy. b. Basic experimental plan (regardless of use of radioisotope tracers) (1) Patients will be obtained through the Department of Urology at Colorado General Hospital and Fitzsimons General Hospital. Only those subjects will be chosen who form stones at a relatively rapid rate (at least every 2 months) and who are free of renal infections. A subject so chosen will be kept on a metabolic ward for a 2-week period so that 2 complete 3-day fecal and urine collections can be made. Total Ca, P, Mg and vitamin B6 intake during this period can be estimated from tables of composition or by actual analysis. The total fecal collection for 3 days will be pooled, homogenized and ashed for a determination of its content of calcium, phosphorus and magnesium. A routine urinalysis (pH, sp. gravity, crystals, etc.) will be performed on each 24-0hour urine collection and at least 500 ml will be saved for subsequent analysis. Proposed urinary constituents to be analyzed for are magnesium, calcium, phosphorus, oxalate, citrate, uromucoid, vitamin 36 and xanthurenic acid. After the specimens are received from the 2 balance periods, 420 mg of MgO (250 mg Mg) will be given daily in a single capsule to be taken after supper. Therapy should continue for at least 6 months, during which time the patient's rate of stone formation will be noted. A 24-hour collection of urine will be made every 30 days and the above-mentioned tests will be performed. Patients may be asked to 43 repeat the balance study after 6 months to ascertain any changes in balance or retention of calcium, magnesium or phosphorus as a result of this treatment. (2) Progress to date To date, one patient has been studies completely in terms outlined above, that is, this man has been on magnesium supplements as treatment for his recurrent urolithiasis. He has been on these supplements for 6 months without a recurrence in stone formation; he was then brought back into the hospital and denied the supplements for a period of a month. During both periods, his urinary excretion of calcium, phosphorus, magnesium, oxalate and mucoprotein was determined. A second patient has been given magnesium for 6 months and has shown a favorable response in that he has not formed stones during this period. He recently returned to the hospital for follow-up studies. The findings on magnesium therapy with these two stone-forming patients will be submitted as a manuscript to the Journal of Urology. Additional patients are understudy with the cooperation of Lt. Col. C. A. Moore, M.D. (MC) of Fitzsimons General Hospital, Dr. O. G. Stonington of Colorado General Hospital, and Lt. Col. J. E. Canham, M. D. (MC) of the Metabolic Division of this laboratory. As additional cooperative patients become available, expanded clinical trials as to the effectiveness of this treatment for chronic lithiasis will be undertaken. 44 c. Experimental plan incorporating use of radioactive tracers (Par. 12b(2) The mineral balance studies thus far conducted on the above subjects appear to indicate abnormalities in calcium and magnesium absorption and excretion. However, the balance techniques leave much to be desired from the standpoint of a precise and exacting procedure to give the definitive information necessary for an unequivocal evaluation of small changes that may occur in absorption or excretion. In addition, the method gives little information on turnover rates or retention of the dietary calcium and magnesium and of the magnesium supplements. In order to obtain the desired information that may permit a better understanding of the cause of uroliths in humans and the effect of magnesium in their treatment, the use of Magnesium-28 and Calcium-45 or 47 is proposed. Accurate information on the absorption and turnover of the elements in the stone-forming subject could be readily obtained with the use of these isotopes. The information could be obtained on the patient both before treatment and after a period of magnesium treatment to determine changes or interactions that may have occurred in calcium or magnesium metabolism. Such data may give an insight into the mechanism of action involved. Of equal importance, comparative studies with the use of several salts or oxides of Magnesium-28 could be readily performed in an attempt to explain the reason for the apparent success obtained with MgO at this laboratory, while other salts of magnesium have been of no value (4, 8). Similar studies in a minimum number of normal volunteer subjects would be carried out as necessary to evaluate the findings in the patients. 45 It is hoped that a better understanding of the problem would result from the isotope studies which would lead to a screening test that would identify which stone-forming patients that could be expected to receive beneficial effects from magnesium therapy. The performance of the isotopic studies indicated appear highly necessary before recommendations or large scale treatment with magnesium be initiated with stone-forming subjects. d. Procedures and health physics The patients would be handled in a manner similar to that employed at present for non-radioactive mineral balance studies of calcium, magnesium and phosphorus. Selected stone-forming patients or normal volunteers would be placed on the Metabolic Ward at this laboratory. The subjects would receive a controlled diet without magnesium therapy. After a period of 7-10 days on these diets, with balance studies conducted, the patients would receive a tracer dose of either Mg-28 (not exceeding 20 uc) orally. Markers would be employed to assist in the stool collections. Urine, stools and blood samples would be collected and analyzed until essentially no activity could be detected. It is hoped that dosages of radioisotope may be reduced further to one-half the amounts indicated and still permit satisfactory measurements. This would then permit double labeling of selected patients or repeat labeling of a patient or 46 normal volunteer following 6 months of magnesium therapy without approaching the maximum permissible body burden of radiation. If the dosage cannot be reduced sufficiently, then other patients or subjects would receive the second isotope or the isotope after 6 months of magnesium therapy. Balance studies with known diets and intakes and maintenance on the Metabolic Ward for periods of 5-6 days (i.e. before and after the 6-months' therapy period--not maintenance on the Metabolic Ward throughout the 6-months' period) would be associated with all subjects. If the oral studies indicate further evaluation of the retention and turnover of magnesium or calcium in the body, a limited number of select volunteer patients or normal volunteers would receive intravenously administered isotopes. In all instances, the intravenous dose would not exceed 35 uc. For comparative purposes, a limited number (3-5) of normal, healthy volunteer subjects would be place don the same diets and balance studies performed with the use of the same isotopes in the same dosage as employed with the volunteer patients. The healthy physics of Mg-28, Ca-47 and Ca-45 has been considered briefly before. It should be emphasized that the maximum dosages obtained with the amount of isotopes used in the proposed studies will at no time equal the maximum permissible dosage. With the aid of the ICRP Handbook (Appendix I, reference 1), the calculations below were made. In each case "1" 47 is the infinite dose received by the critical organ and "2" is the dose received during the first 13 weeks by the critical organ. The critical organ is given in parentheses. A. 5 uc of Ca-47 administered orally (bone) 1. 0.359 rem 2. 0.359 rem B. 2.5 uc of Ca-47 administered intravenously (bone) 1. 0.150 rem 2. 0.150 rem C. 15 uc of Ca-45 administered orally (bone) 1. 5.883 rem 2. 1.897 rem D. 7.5 uc of Ca-45 administered intravenously (bone) 1. 0.245 rem 2. 0.079 rem *E. 20 uc Mg-28 administered orally (bone) 1. 0.465 rem 2. 0.465 rem *F. 35 uc of Mg-28 administered intravenously (bone) 1. 1.992 rem 2. 1.992 rem *G. 35 uc of Mg-28 administered intravenously (whole body) 1. 0.044 rem 2. 0.044 rem *H. 20 uc of Mg-28 administered orally (stomach) 48 1. 0.116 rem 2. 0.116 rem (i.e., residence time of 1 hour) *I. 20 uc of Mg-28 administered orally (small intestine) 1. 0.079 rem 2. Same (i.e. residence time = 4 hours) *J. 20 uc of Mg-28 administered orally (upper large intestine) 1. 1.284 rem 2. Same (i.e. residence = 8 hours) *K. 20 uc of Mg-28 administered orally (lower largeintestine) 1. 1.720 rem 2. Same (i.e. residence time = 18 hours) *Radiation burden for Mg-28 were obtained by calculations and the use of: 1. ICRP Handbook (Appendix I, reference 1). 2. Peaceful Uses of Atomic Energy, Vol. 24, Part 1, "Isotopes in Biochemistry and Physiology," 1958, United Nations Publication. 3. Radioactive Isotopes in Medicine and Biology: Medicine, S. Silver, 1962, Lea and Febiger, Publishers. 4. The Role of Magnesium in Biologic Processes, J. K. Aikawa, 1963, C. C. Thomas, Publisher. 5. Silver, L., Robertson, J.S. and Dahl, L. K.: Magnesium Turnover in the Human Studies with Mg-28. J. Clin. Investigation 39: 420, 1960. 6. Radiological Health Handbook, PB 121784R, U. S. Dept. of Health, Education and Welfare, Public Health Service, 49 U. S. Dept. of Commerce, 1960. According to information supplied from the above sources, absorption of magnesium from the G.I. tract is very low. However; for the purpose of these calculations, 60% absorption was assumed when the isotope was assumed. As previously stated, elimination after absorption is very rapid. Again, however, in order not to underestimate the dosage, an intake and retention of 90% of the isotope was assumed to be removed from the blood by the critical organ (bone). All collected excreta would be disposed of in an acceptable manner under the supervision of the Radioisotope Branch of this laboratory. e. Personnel H.E. Sauberlich, Ph.D. (PL-313), USAMRNL, Project Leader Lt. Col. C.A. Moore, M.D. (MC), Fitzsimons General Hospital, Project Leader (Also attending physician) Lt. Col. J. E. Canham, M.D. (MC) USAMRNL (Also attending physician) O.G. Stonington, M.D., Colorado General Hospital (Also attending physician) Capt. G. E. Bruce, Ph.D. (Consultant, Tripler General Hospital, Hawaii) Lt. Col. M. E. McDowell, M.D. (MC) (Also attending physician) f. References (1) Chute, R., "Urinary Stone: its Nature and Treatment," The Medical Clinics of North America, Philadelphia, W. B. Saunders, Co., 1958, p. 1427. (2) Grossman, W. The current urinary stone wave 50 in Central Europe. The Brit. J. Urol. 10: 46, 1938. (3) Boyce, W. H. and J. S. King, Jr. Effects of high calcium intakes on urine--human beings. Fed. Proc., December 1959. (4) Boyce, W. H. Nutrition and the formation of urinary calculi. Borden's Rev. of Nutrition Rsch. 21: 27, 1960. (5) Bunce, G. E., P. G. Reeves, T. S. Oba and H. E. Sauberlich. Influence of the dietary protein level on the magnesium requirement. J. Nutrition 79: 220, 1963. (6) Union of Burma Nutrition Report. A report of the ICNND, May 1963. (7) U. S. Army Med. Rsch. and Nutrition Lab. Annual Research Progress Report, June 1963. (8) Boyce, W. H., C. M. Norfleet and F. K. Garvey. Therapeutic approach to the "Problem Patient" with urinary calculi. S. Med. J. 52: 443, 1959. (8) Biological Studies on Calcium, Strontium, Lanthanum and Yttrium. D. Laszlo, p. 62, Peaceful Uses of Atomic Energy 10, 1956, United Nations Publication. 51 Appendix I. References on General Health Physics 1. Recommendations of the International Commission on Radiological Protection, ICRP Publication 2, Report of Committee II on Permissible Dose for Internal Radiation, 1959, Pergamon Press. 2. Radiological Health Handbook, U. S. Department of Health, Education and Welfare, Sept. 1960. 3. Radioactive Isotopes in Medicine and Biology: Medicine, S. Silver, 1962, Lea and Febiger, Publishers. 4. Radioactive Isotopes in Medicine and Biology: Basic Physics and Instrumentation, E. Quimby and S. Feitelberg, 1963, Lea and Febiger, Publishers. 5. Use of Radioisotopes in Animal Biology and the Medical Sciences, Vol. 1 and 2, 1962, Academic Press. 6. Maximum Permissible Amounts of Radioisotopes in the Human Body and Maximum Permissible Concentrations in Air and Water. Handbook 52, U. S. Dept. of Commerce. 7. Progress in Nuclear Energy: Series VI, Biological Sciences, J. G. Bugher, J. Coursaget and J. F. Loutit, Editors, 1959, Pergamon Press. 8. Maximum Permissible Body Burdens and Maximum Permissible Concentrations of Radionuclides in Air and in Water for Occupational Exposure. Handbook 69, U. S. Dept. of Commerce. 9. Progress in Nuclear Energy: Series VII, Medial Sciences, J. G. Bugher, J. Coursaget and J. F. Loutit, Editors, 1959, Pergamon Press. 52 10. Peaceful Uses of Atomic Energy: Vol. 22, "Biological Effects of Radiation," 1958; United Nations Publication. 11. Peaceful Uses of Atomic Energy: Vol. 24, Part 1, "Isotopes in Biochemistry and Physiology," 1958; United Nations Publication. 12. Radioisotope Studies of Fatty Acid Metabolism, J. F. Mead and D. R. Hawton, 1960, Pergamon Press. 13. Peaceful Uses of Atomic Energy: Vol. 10, "Radioactive Isotopes and Nuclear Radiations in Medicine," 1956, United Nations Publication. 14. Clinical Use of Radioisotopes. W. H. Beierwaltes, P. C. Johnson and A. J. Solari, 1957, W. B. Saunders Co., Publishers. 15. The Use of Isotopes in Nutrition Research with Special Reference to Tritium. J. Dane and P. R. Payne. World Review of Nutrition and Dietetics: Vol. 1, p. 207, 1959, Hafner Publishing Co. 53