ATTACHMENT 18 At the request of Col. S. Warren a meeting was held in Rochester, N.Y., Sept. 5, 1945, to work out a detailed plan for a "product" tracer study. The study is to be a cooperative work between the Rochester and Santa Fe Groups. This report is the revised plan resulting from the meeting. Those present at the meeting were Drs. Doudy, Bale, Fink, McKann, Bassett, Hodge and others representing the Rochester group, Capt. Bryan representing Col. Warren's office and W. Langham representing the Santa Fe group. The plan is by no means set. It is merely a result of the meeting in Rochester and of numerous conversations with Col. Warren, Col. Friedall, and Dr. L. H. Hempelmann. The plan is subject to change by any of the persons concerned. Much of the responsibility for details falls entirely upon the Rochester group. These details are not given in this report. W. Langham i I. General Plan 1 II. Detailed Considerations: 2 A. Selection of Subjects 2 B. Control Period (14 days) 2 1. Clinical Observations 2 2. Hematology 3 3. Clinical Chemistry 3 a. Blood 3 b. Renal Function 3 c. Liver Function 3 4. Standardization of Subject 3 5. Standardization of Sampling Procedures 4 6. Collection of Blank Samples 4 a. Blood 4 b. Urine 4 c. Feces 5 C. Experimental Period 5 1. Injection 5 a. Size of Dose 5 b. Nature of Dose 5 c. Preparation of Solution 5 (1) Stock Solution of Plus Four Nitrate 5 (2) Preparation of Injection Solution 6 d. Injection Procedure 7 e. Calibration of Syringe and Determination of Dosage Injected 7 ii C. Experimental Period (continued) 2. Clinical Observations 8 3. Hematology 8 4. Clinical Chemistry 8 5. Collection and Handling of Samples 9 a. Blood 9 (1) Collection 9 (2) Preserving 9 (3) Labeling and Shipping 10 b. Urine 10 (1) Collection 10 (2) Preserving 11 (3) Labeling and Shipping 11 c. Feces 12 (1) Collection 12 (2) Preserving 12 (3) Labeling and Shipping 12 D. Methods of "Product" Analysis 13 1. Blood 13 2. Urine 15 3. Feces 16 E. Records and Correspondence 16 iii REVISED PLAN OF "PRODUCT" PART OF ROCHESTER EXPERIMENT As a result of the Rochester meeting held September 5, 1945, it is now possible to plan the "product" part of the Rochester experiment more or less completely. I. GENERAL PLAN: The general plan of the experiment is essentially unchanged as a result of the meeting. In general, the plan is as follows: Two subjects are to be selected by the Rochester group. These subjects are to be admitted to the metabolism ward for a period of approximately six weeks. Because of general difficulties in getting the metabolism ward staffed and in operation it was decided to admit only two patients during the first six week period. This will provide better opportunity for working out unforeseen difficulties and for standardizing sampling techniques. During the second and third six-week periods four subjects will be handled simultaneously. The first two weeks of each period is to be a control period. During this time it is necessary to accomplish a number of things, such as, determine the degree of normalcy of the metabolism of the subject, collect blank feces, get the subject on a standard diet, and get ward attendants and subject in the habit of collecting all urine and feces. These and other points will be discussed later under the detailed plan of the control period. At the end of the control period each subject will be given five micrograms of product in a single intravenous injection. For the next 24 days all feces and urine are to be collected according to a precise sampling schedule and periodic blood samples are to be taken. These are to be carefully assayed for "product" by the Santa Fe group. 1 The primary purpose of the experiment is to establish on a statistical number of subjects the relationships existing among such factors as the amount of product in the body, the level of product in the blood, the amount excreted in the urine, the amount excreted in the feces, and the variations of the above with time. This information will greatly increase our knowledge of the metabolism by the human of this hazardous and important material. It will also afford us a statistical basis for diagnosing body internal contamination from the analysis of urine or feces, the obvious purpose of which is to retire workers before they have received harmful amounts of the material. II. DETAILED CONSIDERATIONS: A. Selection of Subjects -- The selection of subjects is entirely up to the Rochester group. At the meeting it seemed to be more or less agreed that the subjects might be chronic arthritics or carcinoma patients without primary involvement of bone, liver, blood or kidneys. It is of primary importance that the subjects have relatively normal kidney and liver function, as it is desirable to obtain a metabolic picture comparable to that of an active worker. Undoubtedly the selection of subjects will be greatly influenced by what is available. The above points, however, should be kept in mind. B. Control Period -- At the meeting it was agreed that the control period should last for 14 days. Because of the importance of this period there is no reason why it should not be exceeded if Dr. McKann and Dr. Bassett think it necessary. During this period the following points should receive consideration. 1. Clinical Observations and Records -- Such observations and records are entirely up to the Rochester group. The observations should be those considered necessary by the medical staff. 2 2. Hematology -- If approximately 60 percent of the injected product deposits in the bone, as indicated by rat studies, there may be some manifestation in the blood elements. A complete blood picture should be established during the control period. The exact nature of the hematological investigations and their execution is up to the Rochester group. 3. Clinical Chemistry -- The clinical chemistry is the responsibility of the Rochester group. During the control period all studies should be made that are essential to determining the state of of the bone, liver, and kidney function of the subject. These fall into the following classification: a. Blood Tests -- Usual clinical blood chemistry and any other studies the Rochester group think necessary. b. Renal Function Tests -- Urea clearance and other tests the Rochester group think necessary. c. Liver Function Tests -- Those tests should be made that the Rochester group think necessary to establish the status of the subjects' liver function. The protoporhyria studies of Schwartz are a possibility. 4. Standardization of Subject -- The control period will afford an opportunity to get the subject on a standard diet and on a standard fluid intake. The standardization period should help regulate the subjects' fecal output. If possible the urinary output should be regulated to about 800-1600 ml. per day. 3 5. Standardization of Sampling Procedures -- During the control period it will be possible to determine whether or not the carmine or charcoal marking method can be used for separating the feces into 4-day periods. This will certainly be the preferred method if the patients' bowel movement is adapted to its use. As an alternative measure it may be desirable during this period to establish the average weight of fecal output per four-day period for each subject. If this weight-time relationship can be established it would afford another means of correlating fecal output of "product" with time. This period will also afford the opportunity for patients and ward attendants to establish routine sampling procedure for both feces and urine. 6. Collection of Blank Samples -- Because of the normal radioactivity content of the human body it is necessary to have a few blank samples of feces, urine, and blood from each subject before the injection. a. Blood -- During the control period at least three 15 ml blood samples should be taken from each subject and shipped to the Santa Fe group for blank determinations. These samples should be at least 15 ml in volume. The exact volume should be specified on the label. Sodium citrate should be used as an anti-coagulant and formaldehyde should be added as a preservative. Hemolysis of the sample does not matter. b. Urine -- At least three, and preferably five, 24-hour urine samples should be collected from each subject during the control period. These should be labeled and shipped to the Santa Fe group. This group will use them to establish normal blank values for each subject. 4 Labeling, preserving and shipping of samples are discussed under "Collection and Handling of Samples". c. Feces -- Two four-day feces blanks should be collected during the control period. This will afford an opportunity to test the carmine feeding method of dividing the feces. C. Experimental Period -- At the close of the control period each subject is to receive a single intravenous injection of "product". The next 24 days after the injection will constitute the experimental period. During this time periodic blood samples are to be taken, all urine collected on a 24-hour sampling schedule and all feces collected as individual stools during the first four days and on a four-day sampling schedule thereafter until the end of the experiment. The details of the experimental period are as follows: 1. Injection: a. Size of dose -- The size of the dose is to be 5 micrograms for each subject. b. Nature of dose -- The dose is to be plus four "product" complexed with citrate. The product will be in 0.5 ml of sterile 0.5 percent Na3C6H5O7 * 5-1/2 H2O at a pH 6. c. Preparation of Solution: (1) Stock Solution of Plus Four Nitrate -- The stock solution will be prepared at Santa Fe by W. Langham as follows: Clean by electrolytic polishing a 20 mg piece of pure product metal. Place in a dry 4 ml vol. flask and dissolve in the smallest amount of 6N 5 HCl by adding the acid dropwise: when dissolved add 1.0 ml of 8N HNO3 and warm under a heat lamp until the blue color of the plus three valence state changes completely to the brownish green color of plus four nitrate. Dilute to 4 ml with distilled water. This gives a stable solution of plus four nitrate containing 5 mg of product per ml in 2N HNO3. The exact "product" content of this solution is then determined by radioactivity assay. Two ml of the solution is transferred to a spectrophotometer cup and a spectrophotometric curve run. Special attention will be given to the regions of plus three and plus six peaks. The spectrophotometric curve will be repeated every time the stock solution is used. (2) Preparation of Injection Solution -- The solution to be inject is to be prepared by Dr. Fink. A volume (determined from the radioactivity assay) of the stock solution equivalent to 250 micrograms of "product" is carefully pipetted into a sterile 25 ml vol. flask containing about 20 ml of sterile 0.5 percent Na3C6H5O7 * 5-1/2 H2O solution. The contents of the flask are then diluted to exactly 25 ml with sterile 0.5 percent citrate solution. An aliquot of this solution should be assayed for "product" before the injection in order to make sure that 0.5 ml contains approximately 5 micrograms. Assay should be by direct alpha count. Assuming "product" to give 140,000 alpha disintegrations per minute per microgram, a 50 percent geometry counter would give 70,000 counts per minute per microgram. 6 Trial preparations of this solution will be made at Santa Fe before the stock solution is sent to Rochester. These trial solutions will be assayed for "product" and the pH will be determined. It would seem desirable to make such trial dilutions and tests at Rochester before the preparation of the actual injection solution. Enough stock solution will be sent to allow for such trials. If the pH of the injection solution is not between 5 - 7 it will be necessary to adjust it, as the citrate complex is not stable at low pH. d. Injection Procedure -- The injections are to be performed by the Rochester group. Col. Warren proposed Lt. Valentine as the one to do the injections. Dr. Fink is to be present at all injections to supervise the calibration tests ("dummy injections") discussed and C. 1. c. One-half ml of the citrate solution containing approximately 5 micrograms of product is to be given to each subject in a single injection into a cubical vein using a dry tuberculin syringe. For obvious reasons extreme care should be taken to make sure the vein has been entered before the material is discharged. e. Calibration of Syringe and Determination of Dosage Injected -- The method of "dummy injections" is to be used to determine the actual amount of material received by the subjects. Using the same syringe and needle used for the injection, the same method of filling the syringe and the same technique used for the injection, the same operator is to inject 0.5 ml of the injection solution into each of five 25 ml volumetric flasks partially filled with 4N HCl. Dr. Fink is to take the flasks and dilute each to volume with 4N HCL. From each flask a suitable aliquot is placed directly on platinum foils and counted in an alpha counter. The average "product" content of the five solutions is taken as the dosage given. 7 The injection solution, the "dummy injection" solutions, syringe and needle and a description of the injection technique is then sent to W. Langham of the Santa Fe group where the "dummy injection" the assays, etc., will be repeated as a check on the dosage value. In this connection a standard platinum foil containing a carefully determined number of product alpha counts is to be prepared at Santa Fe and sent to Dr. Fink as a means of calibrating and correlating the alpha counters at the two places. 2. Clinical Observations -- Though it is extremely unlikely that such a small dosage will produce any clinical symptoms, those observations that the medical group consider necessary should be continued throughout the experimental period. 3. Hematology -- Localization of the material in the bone may produce slight changes in the blood elements. The hematological studies that the Rochester Group consider of interest should continue throughout the experimental period. 4. Clinical Chemistry -- Any clinical chemistry tests may be made that are of interest to the Rochester Group. On the basis of the metabolism of product by the rat about 60 percent of the material may localize in the bone, 0.5 - 1.5 percent in the kidney, 1 - 3 percent in the spleen 8 and 5 - 10 percent in the liver. It is doubtful as to whether or not such small amounts of radiation will produce effects in these organs that can be detected by chemical means. One of the most interesting chemical studies would be the coproporphyrin and protoporphyria investigations of Dr. Schwartz of the Chicago Group. Dr. Schwartz should not be approached, however, without Col. Warren's permission. No chemical investigations should be proposed which will interfere with the delivery of urine and feces samples to the Santa Fe group. Occasionally small aliquots of feces and urine samples can be taken for other tests provided the Santa Fe group is informed as to what fraction of a sample was taken or what portion of a particular collection period the aliquot represented. 5. Collection and Handling of Samples:-- a. Blood: (1) Collection -- Four hours after injection a 15 ml blood sample should be taken. The sample should be taken from a cubital vein in the opposite arm from that in which the injection was made. At regular intervals thereafter 15 ml blood samples should be taken. Samples taken at regular two day intervals would be excellent if possible. If the subject does not submit to this frequent sampling, three or four day intervals may be used. The time interval chosen will be left to the Rochester Group. (2) Preserving -- Sodium citrate should be used as an anticoagulant and formaldehyde may be added as a preservative. Do not use oxalate. Hemolysis of the cells does not matter. 9 (3) Labeling and Shipping -- Each sample should be labeled in such a manner that there can be no doubt as to the patient, the volume of the sample and the time elapsed since the injection. Immediate shipping of the sample is not necessary. They may be stored and shipped along with the feces and urine samples. Careful sealing and packing is absolutely necessary as part of the trip to the laboratory is by truck. b. Urine: (1) Collection -- Beginning from the time of the injection all urine is collected. It is not necessary that the subject empty his bladder at the time of the injection. The important point is that the timing of the sampling begin at this point. Because of the high "product" excretion rate during the first 24 hours it would be interesting to take two 12 hour samples the first day after which a straight 24 hour sampling schedule is to be maintained for the next 23 days. In case it is necessary to take small urine samples occasionally for other studies, the exact fractional part of the 24 hour sample taken should be noted on the sample label. In case of spillage the fraction of the 24 hour period represented by the amount spilled should be noted on the label. The excretion of "product" is a function of time and not of urine volume. One way of estimating spillage is to record the time of each voiding. If spillage of the next voiding should then occur, the time interval lost can be estimated. 10 Twenty-four urine samples are to be pooled in one-half gallon Mason fruit jars with glass insert tops and rubber seals. Because of gas formation no jar should be filled over three-fourths full. Another jar should be used if the specimen exceeds three-fourths of a jar full. (2) Preserving -- The addition of 15 ml of 40 percent formaldehyde to each jar should serve to preserve the sample. More formaldehyde may be used without harm. (3) Labeling and shipping -- The label on each bottle should be in waterproof ink. It should specify the hour and date of the beginning of the collection period (the end of the one just previous) and the hour and date of the close of the period (the time of last voiding). It is desirable to have the collection as near to a 24 hour period as possible. Still more important however, is to have the label show the exact time interval represented by the sample. The label should bear information relating the sample to the subject. It was agreed that urine samples would be shipped in Mason fruit jars which will not be returned. It was also agreed that samples would not be packed and shipped from the Rochester Med Annex because of contamination possibilities. The jars are to be shipped in specially built wooden cases, each jar packed individually into individual compartments. Packing cases could be returned to Rochester if necessary. 11 c. Feces: (1) Collection -- Individual stools will be collected and analyzed separately during the first four-day period. For the remainder of the experimental period feces will be pooled in four day periods. If possible carmine feeding should be used to divide feces into four-day periods. The time of the first feeding should be at or near the time of the injection. Samples are to be placed in Mason fruit jars for shipping. (2) Preserving -- The method of Stearns (1929) is perhaps as good as any. The stools are transferred to a Mason fruit jar containing enough 6N HCl to keep the material covered. At the end of the collection period, the whole mixture is heated in a water bath until the material is dark brown. The jars should be placed in a cool bath and heated up gradually to avoid danger of cracking. After heating the material should be diluted to a definite volume with HCl, sealed and shaken vigorously. The sample should then be homogeneous and about the consistency of thick cream. A quart Mason jar may be big enough especially for the individual stool collections. The jars should not be filled over three-fourths full. (3) Labeling and Shipping -- The process of labeling and shipping of feces is essentially the same as that for urine. The label should carry all information necessary to relate the sample to the patient, to the time after injection and to the length of the collection period. 12 Urine, feces and blood samples can be shipped in the same packing case if adequately packed. D. Methods of "Product" Analysis: 1. Blood -- The sample as received is transferred to a 300 ml Kjeidahl flask. About 15 ml of conc. HNO3 is used in making the transfer. About 10 ml of 30 percent H2O2 is added and the flask is heated slowly with a Bunson flame. Two pyrex glass beads may be used to minimize bumping. The wet-ashing with HNO3 and H2O2 is continued until only a white ash remains when the sample is taken to dryness. The ash is taken up in 25 ml of 2N HCl. If solution does not occur immediately the flask is heated in a water bath until the solution is clear. If sufficient activity is expected to permit the taking of aliquots the sample is made up to volume in a 50 ml volumetric flask using 2N HCl. Five ml aliquots are transferred to 40 ml centrifuge cones, and 2 drops of 20 percent hydroxylamina are added. After standing 30 minutes 200 micrograms of La+3 are added in a small volume of chloride or nitrate solution. The lanchanum is followed by 0.5 ml of . The LaF3 precipitate is thrown down by centrifuging at 2000 RPM for 15 minutes. The supernatant is decanted off and the precipitate washed once with one ml of . The LaF3 precipitate carrying the "product" is slurred with a small amount of H2O and transferred quantitatively to a platinum plate. The liquid on the plate is slowly evaporated and the residue ignited to a dull red heat over a burner. The plate is then counted in an alpha counter for a period of from 30 minutes to four hours depending on the number of counts present. 14 If the expected activity is low, the entire ash solution is transferred to a 125 ml separatory funnel and the "product" extracted with cupferron and chloroform as follows: The acidity of the solution is adjusted with NH4OH to the bright green end point of methyl violet (pH ~ 0.4). One and one-half ml of 6 percent aqueous cupferron solution is added and the separatory funnel shaken for one minute. Two ml of CHCl3 are added and the funnel is again shaken for one minute. The CHCl3 phase is drawn off into a 40 = centrifuge conc. The cupferron CHCl3 extraction of the aqueous phase is repeated until the CHCl3 phase is colorless. Usually 4 or 5 extraction are sufficient. The CHCl3 phases are combined in the 40 ml centrifuge tube and evaporates to dryness-in a water bath at about 70C. The residue in the tube is treated with one ml of conc. HNO3 and allowed to remain in the water bath at 70C. for 15-20 minutes. At the end of this time, one ml of 72 percent HClO4 is added and the mixture placed in an oil bath at about 115C. The temperature of the oil bath is allowed to rise gradually to 180C. over a period of about one hour. At the end of this digestion period about one ml of a pale yellow solution remains which becomes colorless on cooling. The cool solution is diluted to 4 ml with distilled water. Two drops of 20 percent hydroxylamina are added and the solution allowed to stand for one-half hour. A LaF3 precipitation is then carried out in exactly the same manner as described previously for high count blot samples. 14 Because the number of counts is usually quite low the plates are counted in a low background Simpson type proportional alpha counter to a 2-4 hour period. Results are reported in counts per minute per 100 ml of whole blood. 2. Urine -- The entire 24-hour urine sample is transferred to a 3 liter orlenmeyer flask and placed in a drying oven at 150-170C. An air inlet tube is inserted through the vent of the oven and into the neck of the flask. A rapid current of filtered air is blown into the flask. By this procedure 1500 ml of urine can be evaporated to dryness in 4 - hours. When the sample is down to dryness the brown residue is taken up in about 100 ml of aqua regia, the aqua regia being used first to wash out the original sample jar. The aqua regia solution is transferred to a 500 ml porcelain casserole and evaporated to dryness on a hot plate. A speedy-vap cover glass is used to cover the casserole. The sample is then wet-ashed by evaporating down several times with conc. ENO3, the ENO3 being used first to wash out the original sample jar and the 3 liter orlenmeyer flask before being poured into the casserole. When evapors to dryness with HNO3 leaves only a yellow salt the material is ignited over a Mecker burner for a few minutes. The white ash remaining after the ignition is taken up in 60 ml of 2N CHl by adding it a few ml at a time, heating and transferring to 200 ml centrifuge bottle with a transfer pipet. A faint turbidity in the solution can be ignored. 15 The dilute HCl solution consisting of 60-80 ml is treated with an excess of conc. NH4OH, the hydroxide precipitate carrying the "product" is centrifuged down and the supernatant decanted. The precipitate is dissolved in about 25 ml of 2N HCl and transferred to a 125 ml separatory funnel, and the centrifuge bottle is washed with two small portions of 0.2N HCl. One mg of added as FeCl3 in 0.2 ml of 0.2N HCl. The solution is neutralized by adding conc. NH4OH dropping to a point just short of permanent precipitation (if permanent precipitation occurs it is dissolved by adding dilute HCl). Two or three drops of 0.1 percent aqueous methyl violet solution may be used for the neutralization. The end point is a bluish-green color which fades rapidly. Immediately after the neutralization the solution is extracts with cupfarron and the "product" determined in exactly the same manner described previously for low count blood samples. Because the counts are usually quite low, the platinum plates are counted 2-4 hours. Results are reported in counts per minute per 24-h urine sample. 3. Feces -- An aliquot of the homogenized sample representing 1-2 days excretion is transferred to a large taired orcelain casserole and evaporated to dryness on a water bath. The casserole is then placed in a drying oven at 105C. for 48 hours. The aliquot is then weighed to determine the dry weight of the sample. The fecal matter is transferred to a 250 ml porcelain crucible and charred under an infrared lamp. When charred the sample is heated over a Mecker burner until a dark grey ash remains. The dry material remaining in the original casserole is washed into the crucible containing the ash 16 using 10-15 ml of conc. HNO3. The HNO3 is evaporated off and the sample is again heated over a burner. When there is no further evidence of charring and ashing over the burner, the sample is placed in a muffle furnace at 800C. for 8-15 hours. After cooling and weighing about 60 ml of 2N HCl is added to the crucible containing the ash. The slurry is heated under an infrared lamp for about 15 minutes and then transferred to a 200 ml centrifuge bottle. The insoluble portion is centrifuged out and the supernatant is transferred to a volumetric flask of a size such that the final solution will contain 2-4 g. of ash per 100 ml. The insoluble portion is digested almost to dryness with about 10 ml of aqua regia. Twenty-five ml of 6N HCl are added and the portion remaining undissolved is centrifuged out and discarded. The supernatant is added to the volumetric flask and the sample is made up to volume. An aliquot of the ash solution equivalent to 1 - 2 g of feces ash is transferred to a 200 ml centrifuge bottle. The solution is adjusted to a pH of 0.4 to 0.7 using methyl violet indicator (bright green to blue green and point). One ml of 20 percent hydroxylamina hydrochloride is added. The solution is heated for two hours under an infrared lamp to insure complete reduction of the "product" to the plus three state. The pH is readjusted using the same indicator. Twenty-five ml of 10 percent oxalic acid is added. A drop of NH4OH is sometimes necessary to start the precipitation of calcium oxalate which carries the reduced "product". The solution is allowed to stand overnight to insure complete carrying of "product". The precipitate is centrifuged out and washed once with 17 one percent oxalic acid. The oxalate precipitate is dissolved in about 10 ml of fuming HNO3 and the acid destroyed by evaporating the fuming HNO3 solution almost to dryness. The residue is dissolved in 25 ml of 2N HCl and transferred to a 125 ml separatory funnel. Five drops of 20 percent hydroxylamine are added and the solution allowed to stand thirty minutes. One mg of Fe+3 is added and the pH of the solution adjusted to the bright green color of methyl violet. From this point on the "product" is extracted with cupferron and CHCl3 and determined by LaF3 carrying in exactly the same way as described for urine and for low count blood samples. The plates are counted for 2 - 4 hours. Results are reported in counts per minute per 24 hours excretion. E. Records and Correspondence -- The matter of record keeping was discuss at the Rochester meeting and it was decided to hire secretarial personnel at Rochester for this purpose. All data except the "product" content of blood, urine and feces samples will originate at Rochester. Therefore this is the logical place to keep the complete record. The Santa Fe group will keep records of all samples received and the "product" analysis. Periodically the analytical results will be reported to the Rochester Group to be incorporated into the complete record. All correspondence regarding this experiment should be carried on through the office of Dr. William F. Hale, University of Rochester School of Medicine and Dentistry, P.O. Box 287, Crittenden Station, Rochester N.Y. and the office of Dr. L. H. Hempelmann, P.O. Box 1663, Santa Fe, New Mexico. 18