and Vogelstein, 1983). At a specific activity of 3000 Ci/mmol, that 50uCi translates to 16.6 femtomoles of P dNTP being added to the reaction mix, while 50 uCi of aP labeled dNTP at a specific activity of 6000 Ci/mmol will add only 8.3 femtomoles to the reac tion. Unless sufficient unlabeled dntP is added the lower mass of the hotter dNTP solution added might end up slowing the random prime reaction down, giving the resulting probe a lower specific activity than the probe that used the 3000Ci/mmol Label Location on the Compound Consider the reason for using a radioactive molecule. Is the reaction involved in the transferring of the radioactive moiety to a biomolecule, such as a nucleic acid, peptide, or protein? Is the in vivo catabolism of the molecule being studied, perhaps in an ADME(absorption, distribution, metabolism, and excretion) study? Or perhaps the labeled molecule is simply being used as a tracer. For any situation, it,'s worthwhile to consider the following impacts of the label location: First, will the labels location allow the label or the labeled ligand to be incorporated? Next, once incorporated, will it produce the desired result or an unwanted effect? For example, will the label's presence in a nucleic acid probe interfere with the probe's ability to hybridize to its target DNA? The latter issue is also discussed in greater detail in Chapter 14, Nucleic Acid Hybridization. There are some reactions where the location of label is not criti- cal. a thymidine uptake assay is one such case. The labeling will york just as effectively whether the tritium is on the methyl group ng. The Form and Quantity of the radioligand The radionuclide is usually available in different solvents. The two main concerns are the effect (if any) of the solvent on the reaction or assay, and whether the radioactive material will be used quickly or over a long period. For example, a radiolabeled compound supplied in benzene or toluene cannot be added directly to cells or to an enzyme reaction without destroying the biological systems; it must be dried down and brought up in a com- patible solvent. Likewise a compound shipped in simple aqueous solvent might be added directly to the reaction, but might not be Becquerels, divide by 27(27.027): 50 uCi=50 x 10-Ci=(50 10°Ci×3.7×l00 dps/Ci)=1.85×10°dps=1.85×10°Bq= 185MBq.) 146
and Vogelstein, 1983). At a specific activity of 3000 Ci/mmol, that 50mCi translates to 16.6 femtomoles of 32P dNTP being added to the reaction mix, while 50mCi of a 32P labeled dNTP at a specific activity of 6000Ci/mmol will add only 8.3 femtomoles to the reaction. Unless sufficient unlabeled dNTP is added, the lower mass of the hotter dNTP solution added might end up slowing the random prime reaction down, giving the resulting probe a lower specific activity than the probe that used the 3000Ci/mmol material. Label Location on the Compound Consider the reason for using a radioactive molecule. Is the reaction involved in the transferring of the radioactive moiety to a biomolecule, such as a nucleic acid, peptide, or protein? Is the in vivo catabolism of the molecule being studied, perhaps in an ADME (absorption, distribution, metabolism, and excretion) study? Or perhaps the labeled molecule is simply being used as a tracer. For any situation, it’s worthwhile to consider the following impacts of the label location: First, will the label’s location allow the label or the labeled ligand to be incorporated? Next, once incorporated, will it produce the desired result or an unwanted effect? For example, will the label’s presence in a nucleic acid probe interfere with the probe’s ability to hybridize to its target DNA? The latter issue is also discussed in greater detail in Chapter 14, “Nucleic Acid Hybridization.” There are some reactions where the location of label is not critical. A thymidine uptake assay is one such case. The labeling will work just as effectively whether the tritium is on the methyl group or on the ring. The Form and Quantity of the Radioligand The radionuclide is usually available in different solvents. The two main concerns are the effect (if any) of the solvent on the reaction or assay, and whether the radioactive material will be used quickly or over a long period. For example, a radiolabeled compound supplied in benzene or toluene cannot be added directly to cells or to an enzyme reaction without destroying the biological systems; it must be dried down and brought up in a compatible solvent. Likewise a compound shipped in simple aqueous solvent might be added directly to the reaction, but might not be 146 Volny Jr. Becquerels, divide by 27 (27.027): 50mCi = 50 ¥ 10-6Ci = (50 ¥ 10-6Ci ¥ 3.7 ¥ 1010dps/Ci) = 1.85 ¥ 106dps = 1.85 ¥ 106Bq = 1.85MBq.)
the best solvent for long-term storage. From a manufacturing per spective, the radiochemical is supplied in a solvent that is a com promise between the stability and solubility of the compound and the investigators convenience. Some common solvents to consider, and the reasons they are used Ethanol, 2%. Added to aqueous solvents where it acts as free radical scavenger and will extend the shelf life of the radio- labeled compound Toluene or benzene. Most often used to increase stability of the radiolabeled compound, and increase solubility of nonpolar ds, such as lipids B-mercaptoethanoL, 5mM. Helps to minimize the release of radioactive sulfur from amino acids and nucleotides in the form of sulfoxides and other volatile molecules Colored dyes Added for the investigator's convenience to visualize the presence of the radioactivity When not in use. the "stock" solution of the radioactive compound is capped and usually refrigerated to minimize olatilization/evaporation What Quantity of Radioactivity Should You Purchase? There are three things to consider when deciding how much aterial to purchase 1. How much activity (radioactivity) will be used and over what period? 2. What are the institutional limits affecting the amounts of radioisotope chosen that your lab may be authorized to 3. What are the decomposition rate of your radiolabeled compound and its half-life. n general you will want to purchase as large a quantity as possible to save on initial cost, while at the same time not com promising the quality of the results of the research by using decomposed material. For example, certain forms of tritiated hymidine can have radiolytic decomposition rates(thymidine degradation) of 4% per week. This decomposition rate is not to be confused with tritium's decay rate, or half-life, which is over 12 years. Stocking up on such rapidly decomposing material, or by using it for more than just a few months could compromise experiments carried out later in the products life. Working Safely with Radioactive Materials 147
the best solvent for long-term storage. From a manufacturing perspective, the radiochemical is supplied in a solvent that is a compromise between the stability and solubility of the compound and the investigator’s convenience. Some common solvents to consider, and the reasons they are used: • Ethanol, 2%. Added to aqueous solvents where it acts as a free radical scavenger and will extend the shelf life of the radiolabeled compound. • Toluene or benzene. Most often used to increase stability of the radiolabeled compound, and increase solubility of nonpolar compounds, such as lipids. • 2-mercaptoethanol, 5 mM. Helps to minimize the release of radioactive sulfur from amino acids and nucleotides in the form of sulfoxides and other volatile molecules. • Colored dyes. Added for the investigator’s convenience to visualize the presence of the radioactivity. When not in use, the “stock” solution of the radioactive compound is capped and usually refrigerated to minimize volatilization/evaporation. What Quantity of Radioactivity Should You Purchase? There are three things to consider when deciding how much material to purchase: 1. How much activity (radioactivity) will be used and over what period? 2. What are the institutional limits affecting the amounts of radioisotope chosen that your lab may be authorized to use? 3. What are the decomposition rate of your radiolabeled compound and its half-life. In general you will want to purchase as large a quantity as possible to save on initial cost, while at the same time not compromising the quality of the results of the research by using decomposed material. For example, certain forms of tritiated thymidine can have radiolytic decomposition rates (thymidine degradation) of 4% per week. This decomposition rate is not to be confused with tritium’s decay rate, or half-life, which is over 12 years. Stocking up on such rapidly decomposing material, or by using it for more than just a few months could compromise experiments carried out later in the product’s life. Working Safely with Radioactive Materials 147
When Should You order the materia? Analvsis Date Ideally you will want to schedule your experiments and your adiochemical shipments such that the material arrives at its maximum level of activity and lowest level of decomposition. This will tend to be when the product is newer, or nearer its analy date(the date on which the compound passes quality control tests and is diluted appropriately so that the radioactive concentration and specific activity will be as those stated on the reference date) Some isotopes and radiochemicals decompose slowly, so it is not always necessary to take this suggestion to the extreme. As you use a radiolabeled product, you' ll come to know how long you can use it in your work. AnI labeled ligand will not last as long as aC labeled sugar. An inorganic radiolabeled compound, such as Na I or sodium chromate, will decompose at the isotopes rate of decay, whereas a labeled organic compound, such as the tritiated thymidine alluded to earlier, will decompose at a much faster rate than the half-life of the isotope would indicate Manufacturers take this into account by having a terminal sale date The material will only be sold for so long before it is removed from its stores. Up until this date you will be able to purchase the material and still expect to use it over a reasonable period of time Reference Date The reference date is the day on which you will have the stated amount of material. If you purchased a 1 mCi vial of 3 P dCTP, yo will have greater than l mCi (37 MBq) prior to the reference date, l mCi on that date, and successively less beyond the reference date. (Note that since you will most likely receive your radioac tive material prior to reference, it is possible to exceed possession limits; consider this when determining limits on your radiation license ) In the case of longer-lived radioisotopes, such as H and C, the analysis date will also serve as the reference date How Do you calculate the amount of emaining Radiolabel? is to use the following exponential decay equation. For conve nience's sake, most manufacturers of radiochemicals provide decay charts in their catalogs for commonly used isotopes. This equation comes in handy for the less common isotopes. A= Aoe 0.693tT 148
When Should You Order the Material? Analysis Date Ideally you will want to schedule your experiments and your radiochemical shipments such that the material arrives at its maximum level of activity and lowest level of decomposition. This will tend to be when the product is newer, or nearer its analysis date (the date on which the compound passes quality control tests and is diluted appropriately so that the radioactive concentration and specific activity will be as those stated on the reference date). Some isotopes and radiochemicals decompose slowly, so it is not always necessary to take this suggestion to the extreme. As you use a radiolabeled product, you’ll come to know how long you can use it in your work. An 125I labeled ligand will not last as long as a 14C labeled sugar. An inorganic radiolabeled compound, such as Na125I or sodium 51chromate, will decompose at the isotope’s rate of decay, whereas a labeled organic compound, such as the tritiated thymidine alluded to earlier, will decompose at a much faster rate than the half-life of the isotope would indicate. Manufacturers take this into account by having a terminal sale date.The material will only be sold for so long before it is removed from its stores. Up until this date you will be able to purchase the material and still expect to use it over a reasonable period of time. Reference Date The reference date is the day on which you will have the stated amount of material. If you purchased a 1 mCi vial of 32P dCTP, you will have greater than 1 mCi (37MBq) prior to the reference date, 1mCi on that date, and successively less beyond the reference date. (Note that since you will most likely receive your radioactive material prior to reference, it is possible to exceed possession limits; consider this when determining limits on your radiation license.) In the case of longer-lived radioisotopes, such as 3 H and 14C, the analysis date will also serve as the reference date. How Do You Calculate the Amount of Remaining Radiolabel? The most straightforward way of calculating radioactive decay is to use the following exponential decay equation. For convenience’s sake, most manufacturers of radiochemicals provide decay charts in their catalogs for commonly used isotopes. This equation comes in handy for the less common isotopes. A = A0e-0.693t/T 148 Volny Jr
where Ao is the radioactivity at reference date t is the time between reference date and the time you are cal culating for, Tis the half-life of the isotope(note that both t and T must have the same units of time) It is easy to use the aforementioned decay charts as shown in the following two examples. Say you had 250uCi of>>s methionine at a certain reference date and the radioactive concentration was 15mCi/ml. Now it is 25 days after that reference date You calculate your new radioac tive concentration and total activity in the vial by looking on the chart to locate the fraction under the column and row that corre- sponds to 25 days postreference. This number should be 0.820 Multiply your starting radioactive concentration by this fraction to obtain the new radioactive concentration: 15mCi/ml x0.820=12.3 mCi/ml The total amount of activity can be likewise calculated for S with a half-life of 87 4 days; namely A Age- 15exp(0.693×25/874)=123mCi/m For the second example you can find out how much activity you had before the reference date. Some decay charts only have postreference fractions, but if you have a l mCi vial of P dUTP at 10mCi/ml, and it is 5 days prior to the reference date, how do you figure out how much you have? Go to the column and row on the P decay chart corresponding to 5 days postreference There you will see the fraction 0.872. You will divide your ref- erence activity and radioactive concentration by this number to obtain the proper amount of activity present, or 1/0.872 1.15 mCi. Note that the values should be greater than the stated amounts of activity and the referenced radioactive concentration For the calculation method you are now looking for Ao. Therefore Ao= Aeos=10exp(0.693 x 5//25)=11.5mCi/ml, using a half-life of 25 days for> P How Long after the Reference Date Can You Use Your material? Radioactively labeled compounds do not suddenly go bad after the reference date. It isnt an expiration date. It is used as a benchmark by which you can anchor your decay calculations Working Safely with Radioactive Materials 149
where A0 is the radioactivity at reference date, t is the time between reference date and the time you are calculating for, T is the half-life of the isotope (note that both t and T must have the same units of time). It is easy to use the aforementioned decay charts as shown in the following two examples. Say you had 250mCi of 35S methionine at a certain reference date, and the radioactive concentration was 15mCi/ml. Now it is 25 days after that reference date. You calculate your new radioactive concentration and total activity in the vial by looking on the chart to locate the fraction under the column and row that corresponds to 25 days postreference. This number should be 0.820. Multiply your starting radioactive concentration by this fraction to obtain the new radioactive concentration: 15 mCi/ml ¥ 0.820 = 12.3 mCi/ml The total amount of activity can be likewise calculated for 35S with a half-life of 87.4 days; namely A = A0e-0.693t/T = 15 exp(-0.693 ¥ 25/87.4) = 12.3mCi/ml. For the second example you can find out how much activity you had before the reference date. Some decay charts only have postreference fractions, but if you have a 1 mCi vial of 33P dUTP at 10 mCi/ml, and it is 5 days prior to the reference date, how do you figure out how much you have? Go to the column and row on the 33P decay chart corresponding to 5 days postreference. There you will see the fraction 0.872. You will divide your reference activity and radioactive concentration by this number to obtain the proper amount of activity present, or 1/0.872 = 1.15 mCi. Note that the values should be greater than the stated amounts of activity and the referenced radioactive concentration. For the calculation method you are now looking for A0. Therefore A0 = Ae0.693t/T = 10exp(0.693 ¥ 5/25) = 11.5mCi/ml, using a half-life of 25 days for 33P. How Long after the Reference Date Can You Use Your Material? Radioactively labeled compounds do not suddenly go bad after the reference date. It isn’t an expiration date. It is used as a benchmark by which you can anchor your decay calculations as described above. Working Safely with Radioactive Materials 149