ATTACHMENT 17 Detailed Plan of Product part of Rochester Experiment L. H. Hempelmann and Wright H. Langham 1. General Plans According to Colonel Warren the Product experiment is to be conducted first because of contamination possibilities from the other materials. Ten subjects will be provided by the Rochester area. These subjects will be admitted to the metabolism ward in a group of four per month for the first two months. The third month there will be only two. Two of the subjects for one of the other studies may be run simultaneously during the third month. During the first six days blank blood, urine and feces samples will be taken and clinical chemistry and hematological tests run. Careful clinical observations will be made also. This is to be done by the Rochester Group. On the morning of the seventh day each subject will receive a single intravenous injection of product. After the injection careful clinical observations, hematology and clinical chemistry will continue throughout the experimental period of 24 days. The feces from each subject will be collected and pooled into samples representing three day's excretion. All urine will be collected on a strict 24-hour sampling basis. A 10-ml. blood sample will be taken from each subject 4 hours after injection and at regular 3 or 4 day intervals until the end of the experiment. The injections, supervision of the ward, hematology, clinical chemistry, clinical observations and sample collections are to be carried out by the Rochester group. All samples are to be properly labeled, packed and shipped to W. H. Langham, P.O. Box 1663, Santa Fe, H.M. It will be the responsibility of the Santa Fe Group to analyze all samples for Product. II. Specific Considerations A. Hematology - 1. Complete Blood Count 2. Sedimentation Rate 1 3. B. Clinical Chemistry As complete clinical laboratory studies as possible to determine function of liver, kidneys and bone marrow. C. Clinical Observations Those recommended by Rochester Group. D. Preliminary Observation Period and collection of blank samples - This period is to consist of the first six days during which the following should be accomplished: 1. Make preliminary clinical observations. 2. Make preliminary hematological and clinical chemistry tests. 3. Establish sampling routine. 4. Collect at least three 24 hour urine samples from each subject for blank determinations. 5. Collect at least two 10 ml. blood samples from each subject for blank determinations. 6. Collect two feces samples from each subject. Each sample should consist of the pooled excrement representing three days excretion. These are for blank determinations. E. Injection - To be performed in the morning of the seventh day. 1. Size of dose, 1 or 5 micrograms? 2. Nature of dose, plus four product complexed with 0.5% sodium citrate in sterile water, at pH 6.0 and in a vol. of 0.5 ml. 3. Preparation of injection solution. (W. Langham) a. Stock solution -- Carefully clean and weigh 10 mg. of pure product metal. Dissolve in the smallest possible amount of 6N HCl. When completely dissolved add 250 lambda of 8N HNO3 and warm under a heat lamp until the color of the solution changes completely from blue to brown. Dilute to 2 ml. with distilled water. This gives a solution of 2 plus 4 nitrate containing 5 mg. per ml. of product in 1N HNO3. This solution is stable over a long period of time with regard to plus four product. A complete spectrophotometric curve will be run however, each time the solution is used to make sure that the material is entirely plus four. This solution will be assayed carefully for total product concentration. (If thought necessary Langham will come to Rochester to prepare the solution) b. Injection solution -- Fifty lambda (250 ??) of stock solution is carefully measured into a sterile 25 ml. volumetric flask containing about 20 ml. of sterile 0.5% sodium citrate (5H2O) solution. The solution is then diluted to exactly 25 ml. with sterile citrate solution. Test preparations of this solution will be made. These will be assayed carefully for product concentration and pH as well as for the per cent of plus 4 product to make sure no valence disproportination occurs during dilution. In case all concerned agree that the dose should be 1?? instead of 5?? the above solution will be diluted five fold with 0.5% citrate solution. 4. Injection procedure -- The injection is to be performed by the Rochester group. One-half ml. (5.0?? product ?) of the citrate solution is given to each subject in a single injection into a cubital vein using a dry tuberculin syringe. Care should be taken to avoid any leakage into the tissue. 5. Calibration of syringe and determination of amount of product injected- Using the same syringe and needle and the same technique used for filling the syringe and making the injection; 0.5 ml. of the injection solution is discharged into each of five twenty-five ml. volumetric flasks containing 4N HCl. These will be diluted to volume with 4N acid and mixed thoroughly. 3 If the Rochester Group is not equipped to do product assays, these flasks should be carefully sealed and shipped to the Santa Fe Group. One hundred lambda portions of each solution are plated directly on platinum plates and counted in an alpha counter. The average product content of these five solutions is taken as the dosage received by each subject. III. Collection and handling of samples - 1. Blood samples: a. Collection - a 10 ml. blood sample should be taken from each subject at regular intervals perhaps every 3 or 4 days. The first sample should be taken 4 hours after injection. b. Preserving - The sample should be sealed in a suitable tube containing sodium citrate as an anticoagulant and one ml. of formaldehyde as a preservative. c. Labeling and shipping - The sample should be labeled with an identification number relating it to the subject and his clinical record. The label should also give the date, and the time elapsed between the time of injection and taking of the sample. The samples should then be adequately packed and shipped. 2. Urine samples: a. Collection: -- Straight twenty-four hour samples are to be taken for the entire observation period. b. Preserving -- Each sample is transferred, as collected, to a suitable bottle (Baxter bottles?) containing 10 ml. of 40% formaldehyde. The formaldehyde should be added at the beginning of the collection period instead of at the end. c. Labeling and Shipping -- The tag on the bottle should carry an identification number relating the sample to the individual and to the clinical record. The label should give the date of the beginning of the collection period, the actual time at which the collection period began and the actual time of the close of the period. It is of greatest importance that any losses be indicated. Therefore, if the time of each voiding is recorded on the label and time of loss indicated, some estimation of the period of loss in hours can be made. The volume lost means nothing, the important thing is the time interval the loss represents. 4 3. Feces samples: a. Collection -- The stools from each subject should be pooled into samples representing three day's excretion. The time of each stool should be noted in order to permit the estimation of the time interval represented by any losses that may occur. b. Preserving -- The jar or container into which the stools are transferred should contain, 100 ml. of 4N HCl as a preservative. The HCl should be added to the container at the beginning of each collection period. At the close of the collection period the container should be closed and the sample thoroughly emulsified with the preservative by vigorous shaking. c. Labeling and Shipping -- The tag on the feces container should carry an identification number relating the sample to the individual and his clinical record. The label should give the date and time of the beginning and the date and time of the close of the collection period. It should also give the time of each stool in order to enable one to estimate the time interval represented by any losses that may occur. IV. Analysis of Samples: This is to be done by the Santa Fe Group using procedures that are already standardized. GENERAL PLAN OF ROCHESTER EXPERIMENT The following is a general picture of the Rochester Experiment as obtained from Colonel Warren during a ten minute conversation. GENERAL PLAN -- A. Fifty subjects are to be provided by the Rochester Area. These subjects are to compose 5 groups of 10 subjects each. Group I will receive product; Group II will receive radium; Group III, postum; Group IV, tuballoy; and Group V will receive lead. Each group will be under observation for 30 days as follows: 1. The first week will be a clinical observation period during which time the status of kidney and liver function will be established. Blanks on feces, blood and urine will be taken and other preliminaries taken care of. 5 2. Injection will be with a single dose, the size of which will be the same for each member of the group. 3. Feces, blood and urine samples will be taken with great care and sent to the various group heads for the purpose of quantitating the amount of the injected material occurring therein. 4. The ward will be under 24-hour supervision. Four subjects will be handled at a time. 5. Clinical observations and studies will be carried on throughout the entire 30 day period. B. Purpose -- The purpose of the study is to establish, on a statistically significant number of subjects, the metabolic behavior of the hazardous materials, product, radium, postum, tubally and lead. More specifically this will serve to establish the relation between any two of these hazardous materials in regard to a number of metabolic processes such as: 1. Rate of urinary and fecal excretion. 2. Ratio between blood level and rate of urinary and/or fecal excretion. 3. Relation between size of dose and blood level. 4. Total body retention of material in relation to time after injection. 5. Relation of blood level to time after injection. 6. The spread in individual variations with regard to blood level, urinary excretion and fecal excretion as a function of size of dosage and time after injection. C. Personnel and Distribution of Responsibility: 1. General Director of Rochester Area, Dr. Andrew Dowdy, Monroe 8972, Rochester, N.Y. 2. In charge of Wards, Dr. William McKann and Dr. Sam Bassett. 3. Injection, Lt. Valentine. 4. Postum, William Bale. 5. Product, W. H. Langham & L. H. Hempelmann. 6 6. Radium -? 7. Tuballoy, Harold Hodge. 8. Lead -? 9. Statistics, Murray Wantman 7