[21]
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DESIGN OF DOUBLE LABEL E X P E R I M E N T S
Acknowledgments We would like to thank the numerous investigators who made unpublished information available to us. This work was supported by Grant No. GM 17239 from the National Institute of General Medical Sciences of the National Institutes of Health and Contract No. 72-3236 within the Special Virus-Cancer Program of the National Cancer Institute of the National Institutes of Health. W. A. S. is a postdoctoral fellow of the United States Public Health Service.
[21] T h e D e s i g n o f D o u b l e L a b e l Radioisotope Experiments
By
EDWIN D. BRANSOME, JR.
Within the context of this volume, it seems reasonable to consider only the limited number of radioisotopes which are usually used as labels for quantities of enzyme, hormone, or nucleotide synthesized, as substrates in systems affected by hormones or cyclic nucleotides, or nucleotides. Table I provides such a limited list. The maximum energies (Emax) involved in their radioisotopic decay are a fair indication of whether one radioisotope in a sample may adequately be determined in the presence of another. The gas ionization (Geiger-Miiller) detectors used in "planchet" counting are not capable of resolving the energies of such isotope pairs. Filters m a y be inserted, however, between sample and detector, quantitatively absorbing emissions of the lower energy radioisotope. This is satisfactory only if there is a wide separation of the two energy spectra, if TABLE I PROPERTIES OF RADIOISOTOPES COMMONLY EMPLOYED
Isotope 3H 14C 3~S 3~p 12~I 131I
Principal emission on decay t~ ~ ~ ~ ~, K-X-ray ~
• Decay by electron capture.
E .... (keV) 18 156 168 1700 35 27" 360 610
Relative abundance (%) 100 100 100 100 7 -80 87
Scintillation detector Liquid Liquid Liquid NaI, liquid NaI, liquid
294
G E N E R A L METHODS FOR EVALUATING HORMONE EF F EC TS
[21]
tritium (~H) with its very low fl energy is not one of the isotopes, and if samples are counted at "infinite thinness"--without significant selfabsorption. Indeed, until the advent of scintillation counters about twenty years ago, double-isotope experimental designs were rarely attempted. Scintillation Counters
Birks 1 has provided a thorough and basic review of the technology involved in "gamma" counters where the detector is usually an activated N a I crystal, which responds to as, ,/s, and K-X-rays and of liquid scintillation counters in which an aromatic phosphor in solution serves as the scintillator. These respond to fls as well as as, ~s, and K-X-rays. In both types of counter, the photoelectron spectra are proportionate to the energy spectra of the photons emitted from radioisotopic decay. " G a m m a " Counters
There is only slight degradation in the resolution of spectra in N a I crystal counters, so that isotope pairs with energies above the crystal's scintillation threshold can be easily separated for counting, with the proviso that overlap of the two spectra be ascertained with standards of the same geometry. Regoeczi and Wehber have recently shown that spillover of 131I into the ~2'~I spectrum varies with the well counter used and the 131I count rate. 2 For example, optimum channels in one of our automatic well counters yield an 125I efficiency of 37.6% and an 1'~I efficiency of 38.8%. The efficiencies depend not only on pulse height analyzer settings, but also upon the shielding and geometry of the crystal and of the counting chamber.) While the spillover of 125I into the ~3lI channel is inconsequential: 0.28%, the ~3~I contribution to the 1'-'5I channel is significant: 9.17%. Therefore 1~I cpm = A - - (0.0917 X B) if A represents the cpm in the ~2"~I channel and B represents cpm in the 1'~I channel. Although "gamma" counting does not involve corrections for complex quenching phenomena as does liquid scintillation counting, self-absorption of emitted energy may be quite significant and may vary with the isotope being counted. The examples of ~-~I and 1~I counting given in Table II should serve as a warning that standards for 7 counting should be as similar in geometry to the unknowns as possible. 1j. B. Birks, "The Theory and Practice of Scintillation Counting." Macmillan, New York, 1964. E. Regoeczi and C. Webber, I. Nucl. Biol. Med. 16, 10 (1972).
[21]
DESIGN OF DOUBLE LABEL E X P E R I M E N T S
295
TABLE II EFFECTS OF SAMPLE GEOMETRY ON RELATIVE DETECTION EFFICIENCY a
Isotope 125I 131I i~sI i~II 14C aH
Aqueous solution
Whatman No. 1 filter paper
Cellulose nitrate filter
A. "Gamma" NaI crystal scintillation counter 1.00 0.92 1.11 1.00 0.85 0.86 B. Liquid scintillation counter 1.00 0.65 1. O0 0.96 1.00 0.71 1.00 0.09
0.82 0.98 0.91 0.42
The counts per minute in a 0.1-ml aqueous solution of [~25I]NaI, [l~lI]NaI, [14C]adenine, or cyclic[SH] AMP were taken as 1.00 and compared to replicates air dried onto discs 20 mm in diameter. Gamma counting was carried out in a Nuclear Chicago automatic gamma system with a 2-inch NaI crystal using optimum channels for each isotope. The samples in 10 X 75 mm Pyrex test tubes were inserted into cellulose nitrate counting tubes. Liquid scintillation counting was with a Beckman LS-150 system with a wide channel encompassing the total spectrum of both isotopes. The aqueous samples were counted in toluene 7.0 g/liter PPO (2,5-diphenyloxazole), 10 % Biosolv BBS-3 (Beckman) solubilizer. The samples dried onto the filters were counted in toluene-PPO without Biosolv. Liquid Scintillation C o u n t i n g In most double-label experiments, two counting channels are used and the photoelectron spectra are only partially separated. In liquid scintillation counting there is too great a loss of detection efficiency if each isotope is eliminated entirely from the other channel: the photoelectron spectra of the fluorescence emitted from the organic phosphors are too broad. Indeed, if the photon energies are not widely separated, it is statistically impractical to eliminate the spillover of either isotope from the other channel. A decrease in detection efficiency can be produced by impurity or chemical quenching which interferes with the excitation of the scintillation phosphor by radioactive photons or by "color" quenchers which absorb the emitted fluorescence and thus interfere with the transmission of light to the photomultiplier tubes. The extent of quenching in a homogeneous solution of sample and phosphor in solvent will cause pulse height shifts of fluorescence spectra to lower photoelectron energies as an inverse function of the energy of the emitted photons. Quenching in samples will thus affect the detection efficiencies of two isotopes variably, the spectral
296
GENERAL
METHODS
FOR
EVALUATING
HORMONE
EFFECTS
[21]
shift of the lower energy isotope being greater. There is then no single set of optimum instrument settings for specific isotope pairs. Methods of quench correction for homogeneous samples (samples in solution or in translucent emulsions of fine micelles) are reviewed thoroughly in recent literature and will therefore not be dwelt upon here. A reading of references cited in footnotes 3-7 should provide a thorough grounding in theory and practice. I have summarized the principal attributes of the three approaches in Table I I I . Correction of the overlap of radioactivities must be carried out before quench correction. The questions t h a t deserve attention in this review are: (a) H o w are the cpm of one isotope mathematically best separated from the cpm of another? (b) H o w are the optimum instrument settings determined for a specific isotope pair? (c) W h a t are the restrictions on sample preparation for statistically reliable double isotope counting? M a t h e m a t i c s o] Double-Isotope Counting. One approach to doubleisotope counting is to continue to use the discriminator settings for single isotopes. Since each channel then counts contributions from both isotopes, simultaneous equations are necessary for the calculation of the absolute radioactivity (dpm) of each sample: H : radioactivity of the isotope H of greater E,la~ in the sample (dpm) L: radioactivity L of lesser E ..... in the sample (dpm) h l : detection efficiency of H in channel 1 (determined by sample channels ratios or external standardization, and expressed as a fraction of 1) h2: detection efficiency of H in channel 2 ll: detection efficiency of L in channel 1 12: detection efficiency of L in channel 2 N I : net cpm (cpm - - background) in channel 1 N2: net cpm in channel 2 H = [N1 - N 2 ( l l / 1 2 ) ] / [ h l - h2(ll/12)]
(1)
L = [N2 - N l ( h 2 / h l ) ] / [ 1 2 - l l ( h 2 / h l ) ]
(2)
3j. B. Birks, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 283-292. Grune & Stratton, New York, 1970. 4C. T. Peng, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 283-292. Grune & Str~tton, New York, 1970. M. P. Neary, and A. L. Budd, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 273-282. Grune & Stratton, New York, 1970. P. D. Klein, and W. J. Eisler, in "Organic Scintillators and Liquid Scintillation Counting" (D. L. Horrocks and C. T. Peng, eds.), pp. 395-418. Academic Press, New York, 1971. 'F. E. L. Ten Haaf, in "Liquid Scintillation Counting" (M. A. Crook, P. Johnson, and B. Scales, eds.), Vol. II, pp. 39-48. Heyden, London, 1972.
[21]
DESIGN OF DOUBLE LABEL EXPERIMENTS
297
TABLE I I I METHODS OF QUENCH CORRECTION IN LIQUID SCINTILLATION COUNTING A. Internal Standardization ("Spiking") Advantages Most accurate of any method if addition of a known standard to the sample is careful and reproducible. Differences in the effects of impurity and color quenching (which may be important with 14C and more energetic isotopes) are obviated Disadvantages The samples must be homogeneous; the standard must be similar chemically to the sample solute if there is any question of an incomplete solution. Handling errors in addition of the standard will be large if the procedure is careless; ideally this procedure should be done in duplicate. Once a sample is "spiked," it cannot be recounted. The procedure is extremely time-consuming. In double-label experiments, it is a laborious and statistically uncertain approach B. Sample Channels Ratio Advantages Data may be obtained in two channels simultaneously; thus, counting the sample only once is sufficient. The sample itself is not altered. Accurate for moderate and high count rates of single isotopes which are quenched slightly to moderately. Independent of sample volume over a wide range. One series of quenched standards will correct for both sorts of quenching of 3H. Nonhomogeneous samples (in suspension, emulsion, on solid supports) may be quench corrected, as long as standard curves are derived from known samples of the same composition and geometry Disadvantages The procedure is inaccurate for highly quenched samples or for samples with low count rates because strong color quenching of 14C or more energetic isotopes is not adequately corrected. With two overlapping isotopes, corrections for spillover must be made first, with a significant additional statistical error C. External Standard Channels Ratios This procedure, involving exposure of the unknown sample to an external 7 source takes advantage of the secondary Compton electron generated within the sample and the resulting broad fluorescence spectrum. Modern instrumentation monitors and subtracts sample cpm from two separate channels and then ratios the Compton cpm; single channel ES is undesirable inasmuch as the dependence of cpm on sample volume is considerable Advantages Application to samples of low activity. Statistically the most suitable for doubleisotope quench correction Disadvantages Least accurate for highly quenched samples in which differences between the photoelectron spectra of sample and Compton electrons are magnified. Samples must be homogeneous. This technique is not applicable to samples in suspension, on solid supports, etc.
298
GENERAL METHODS FOR EVALUATING HOR3IONE EFFECTS
[21]
The advantage to this procedure is that H can be counted at maximum efficiency unless there is severe quenching, but a considerable accumulation of statistical error results from the repeated application of calculated efficiencies and from the relatively complicated algebra. Instead we sacrifice some H efficiency: counting the higher energy isotope in a channel from which L is for all practical purposes excluded. In this case: g = N1/hl
L = IN2 -
(3)
(N1/h2)]/12
(4)
The propagation of error is greatly decreased by such a simplification. In our hands, analysis of the variance occasioned by the use of Eqs. (1) and (2) results in unacceptably large cumulative errors, particularly when count rates for one or both isotopes are low and the overlap is large. There have been several suggestions in the recent literature that simultaneous equations may reliably differentiate two isotopes with considerable spectral overlap. Such advice is dangerous; if simultaneous equations are employed the exclusion of isotope L from channel 1 should still be maximal. Since the effect of quenching on photoelectron spectra tends to this exclusion, such a stipulation is reasonable. The "spillover fraction" (N1 X h2) of Eq. (4) which is a constant in gamma counting, will increase in liquid scintillation counting as the spectrum of isotope H is quenched and progressively more H cpm fall in channel 2. In Beckman scintillation counters with "automatic quench corrections (A.Q.C.)" this fraction can be held relatively constant, s-" but in others, a quench correction curve for this fraction must be obtained, just as it is obtained for the detection efficiencies of the two isotopes in their optimum channels. Occasional investigators have attempted to use simultaneous equations to solve for the activities of three isotopes in the same sample, using two or more channels. The statistical errors implicit in such procedures are without exception unacceptably large. It is sometimes possible to separate one overlapping isotope from another, however, by combining different counting methods. 131I may be separated from 14C or 3~S, and 125I from all, by counting the samples in a gamma counter and then by liquid scintillation alone. It is important that 131I or 12~I efficiency be determined in both counters so that an aecurate "spillover" curve of either iodine isotope into the 14C, 35S, or 3H channel can be calculated from standards. This approach applies Eqs. (3) and (4) to data from two instruments rather than from one. 8 C. H. Wang, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 305-312. Grune & Stratton, New York, 1970. 9 M. F. Grower, and E. D. Bransome, Jr., Anal. Biochern. 31, 159 (1969).
[21]
DESIGN OF DOUBLE LABEL EXPERIMENTS
299
Energetic fls may be also counted in solution in the absence of phosphors by virtue of the Cerenkov effect?° Of the isotopes in Table I, ~P and ~31I can be counted at reasonable efficiencies without any impurity quenching, but Cerenkov photoelectron spectra are so poorly resolved that double isotope counting is not practical. Instrument Settings. The method of selecting discriminator settings for the single-channel counting of a single isotope is straightforward. Since background cpm increase with channel width, the upper potentiometer setting should be decreased to the point where the count rate of a sample of the isotope begins to become significantly diminished. The lower discriminator should be set close to 0 and raised only if there is a major contribution of instrument "noise" to background in the low energy range, a problem not significant in instruments of recent manufacture. In double-label counting there is an obvious conflict between the desires for minimum spillover and maximum efficiency. A general approach to the most effective compromise in any particular situation would obviously be of value. Klein and Eisler H have proposed a performance criterion, P. In the terms of Eqs. (1)-(4) P = hl ×12 X S
(5)
with hl and 12 the detection efficiencies of the two isotopes in their respective channels and S a function of the count ratios between the two channels for each isotope. Comparing a series of settings, however, may be difficult since, as is usually the case, one setting may be superior regarding only one or two of the three factors. Davies and Deterding'-' have introduced a less biased but more complex algebraic approach to find optimum settings, using a weighted sum of the activities of the two isotopes more or less independent of the contribution of background cpm, which I consider a bit too complex to review here. Graphical approaches which are perhaps a bit easier to comprehend are represented in Figs. 1 and 2.13 The procedure for choosing settings for channels for double-isotope counting in an LS counter with logarithmic or pseudologarithmic amplification is illustrated in Fig. 1. The most efficient channel for each isotope is determined separately as outlined above, and the effect of progressively lowering the upper discriminator in 20-division steps is plotted as the lo R. P. Parker, and R. H. Elrick, in " T h e Current Status of Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 110-122. Grune & Statton, New York, 1970. 11p. D. Klein and W. J. Eisler, A~al. Chem. 38, 1453 (1968). 12p. T. Davies and J. H. Deterding, D~t. J. Appl. Rad. Isotopes 23, 293 (1972). laE. D. Bransome, Jr. and S. E. Sharpe, III, Anal. Bioehem. 49, 343 (1972).
300 j
hi Z Z
GENERAL METHODS FOR EVALUATING HORMONE EFFECTS I00
C.;,
80
a_ 0
60
d
o I--
ii::::iiiiiiii ii ~ 40
i!iii::iiiiil::ililiii::ii::ii:j:~:,
0 I--
2: I.Lf L3 r',.-
LLJ n
[21]
20 "
11 I00
200
300
400
500
600
700
800
900
I000
POTENTIOMETER DIVISIONS SETTING AT UPPER DISCRIMINATOR
Fie. 1. Method for selecting channels for double-isotope counting in counting systems with logarithmic or pseudologarithmic amplification. The cpm in cumulative 2% (20 potentiometer divisions) narrow channels are expressed as percentage of total cpm in the most efficient wide channel. The lowest discriminator setting reaching 100% on the ordinate is the upper end-point of the isotope energy spectrum in the specific counter being examined. From E. D. Bransome, Jr. and S. E. Sharpe, III, Anal. Biochem. 49, 343 (1972).
percent of total cpm in the full channel. The H channel chosen should be at the L end point so that there is no spillover of L into the channel used for H. With our Beckman LS-150 (Fig. 1), we found the ~31I channel to be between 260 and 660 discriminator divisions. The upper discriminator setting for the L channel can be decided by identifying the region of maximum difference between the higher and lower energy isotope plots. The shaded area in Fig. 1 indicates the area of proper choice. We chose the 0-260 channel for maximum 125I. A 0 - 1 4 0 channel would have been a better selection if the 131I activity tended to be considerably in excess of that of 1~5I, or if the unknown samples to be counted were considerably quenched so that the 1:5I photoelectron energy spectrum was significantly shifted to the lower end. For "linear" LS counters, the additional requirement of gain adjustment is the most important consideration involved in selecting channels for single- or double-isotope counting. It is not possible to comprehend the complete spectra of more than one isotope by varying the discriminator settings, unless the gain settings are significantly different. The effect of grain on counting efficiency in a wide channel should therefore be systematically examined. To minimize the effect of quenching, the maximum gain for a specific level of efficiency should be chosen so that as much as possible of the spectrum fills the window.
[21]
DESIGN OF DOUBLE LABEL EXPERIMENTS
301
bOO0%ds
Z
W
t J J
I00%.
i l d
f
i f J J i i d ,S 10%-
i J jJ S I
i
S S
S
i
16% iob~o 131I EFFICIENCY
ioOO~o
Fro. 2. Engberg plot of 125I and '31I in a Packard Tri-Carb 3375 LS counter. The efficiencies were determined in a fixed channel at various gain settings and plotted against each other (solid line). The best choices of isotope ratio can be identified by determining the greatest distance of the plotted curve from a 45 ° line drawn from the base (the dashed line). The best 1~1I channel is obvious: the gain with maximum efficiency and no 12~I spillover. For 125I, the best solution is a compromise between low ~31I spillover into the 1-'5I channel, and high 1~I efficiency. Maximal 1~I efficiency is accompanied by more than 20% spillover. If there is a substantial excess of ~1I dpm over 12~I dpm, a lower 1~I efficiency (at higher gain) should be offset by a further decrease in ~ I spillover. From E. D. Bransome, Jr. and S. E. Sharpe, III, Anal. Biochem. 49, 343 (1972). I f the absolute r a d i o a c t i v i t y of the i s o t o p e ( s ) being counted in a s a m p l e is not k n o w n , a comparison of cpm at each gain is sufficient, but there is an a d v a n t a g e to k n o w i n g counting efficiencies. K o b a y a s h i and M a u d s l e y 14 h a v e shown t h a t log-log plots ("Engberg plots") of isotope efficiencies in a fixed w i n d o w can be used to determine the ability of a linear LS counter to separate an isotope pair. Figure 2 shows our results counting 131I and 125I with a P a c k a r d 3375. B e c a u s e the effect of quenching on the lower energy isotope increases the isotope separation, using o n l y l e a s t - q u e n c h e d isotope standards to set up counting channels is a sufficiently rigorous procedure. 14y . Kobayashi and D. V. Maudsley, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 76-85. Grune & Stratton, New York, 1970.
302
GENERAL METHODS FOR EVALUATING HORMONE EFFECTS
[22]
Sample Preparation. Most of the important considerations of sample preparation for liquid scintillation counting can be conveniently reviewed in references cited in footnotes 15 and 16. It is worth reiterating that for calculations of the radioactivity of an isotope pair to be reliable, both isotopes must be uniformly distributed in the sample. Translucent emulsions with microscopic micelles may be counted as well as true solutions if three cautions are observed: (a) If one of the isotope pair is 3H or 1~5I, it must be in the same phase as the other isotope. This might seem to be a trivial problem since emulsion counting is of aqueous samples, but it is not; there may be differential extraction of the labeled solutes into the organic solvent. (b) Absorption (of the lower energy isotope in particular) onto the vial surface may greatly alter detection efficiency.17 (c) If the E .... of an isotope is equivalent to or greater than 14C, and a commercial solubilizer of aqueous samples has been included in the sample, the surfactant itself may fluoresce in response to radioactivity. This renders quench correction curves derived from sealed commercial standards invalid2 s All these proscriptions may be summarized in the cardinal, and frequently neglected, general rule for scintillation counting: standards should be of the same geometry as the unknown samples. 15ff. C. Turner, "Sample Preparation for Liquid Scintillation Counting," Review 6. The Radiochemical Centre, Amersham, England 1967. Reprinted in "Handbook of Radioactive Nuclides" (Y. Wang, ed.) pp. 256-273. Chem. Rubber Publ. Co., Cleveland, Ohio, 1969. '6"The Current Status of Liquid Scintillation Counting" (E. D. Bransome, ed.), Chapters 16-27. Grune & Stratton, New York, 1970. 17G. ft. Litt and H. Carter, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, ed.), pp. 156-163. Grune & Stratton, New York, 1970. ,8 S. E. Sharpe, I I I and E. D. Bransome, Jr., Anal. Biochem. 56, 313 (1973).
[22] U s e o f A n t i b o d i e s t o N u c l e o s i d e s a n d N u c l e o t i d e s in S t u d i e s o f N u c l e i c A c i d s in Cells
By B. F.
ERLANGER, W . Y. KLEIN, JR., V. G. DEV, R. R. SCHRECK,
and 0. J. MILLER The preparation of purine- and pyrimidine-protein conjugates and their use in eliciting base-specific antibodies that react with nucleic acids have been described in this series, Volume 12B [173]. Their reaction with nucleic acids requires that the purine or pyrimidine bases be unpaired, i.e., that the nucleic acid be denatured or, at least, have single-stranded regions. It follows, therefore, that demonstrable reaction with these antibodies is evidence for "single-stranded areas." Fluorescein-tagged
293
DESIGN OF DOUBLE LABEL E X P E R I M E N T S
Acknowledgments We would like to thank the numerous investigators who made unpublished information available to us. This work was supported by Grant No. GM 17239 from the National Institute of General Medical Sciences of the National Institutes of Health and Contract No. 72-3236 within the Special Virus-Cancer Program of the National Cancer Institute of the National Institutes of Health. W. A. S. is a postdoctoral fellow of the United States Public Health Service.
[21] T h e D e s i g n o f D o u b l e L a b e l Radioisotope Experiments
By
EDWIN D. BRANSOME, JR.
Within the context of this volume, it seems reasonable to consider only the limited number of radioisotopes which are usually used as labels for quantities of enzyme, hormone, or nucleotide synthesized, as substrates in systems affected by hormones or cyclic nucleotides, or nucleotides. Table I provides such a limited list. The maximum energies (Emax) involved in their radioisotopic decay are a fair indication of whether one radioisotope in a sample may adequately be determined in the presence of another. The gas ionization (Geiger-Miiller) detectors used in "planchet" counting are not capable of resolving the energies of such isotope pairs. Filters m a y be inserted, however, between sample and detector, quantitatively absorbing emissions of the lower energy radioisotope. This is satisfactory only if there is a wide separation of the two energy spectra, if TABLE I PROPERTIES OF RADIOISOTOPES COMMONLY EMPLOYED
Isotope 3H 14C 3~S 3~p 12~I 131I
Principal emission on decay t~ ~ ~ ~ ~, K-X-ray ~
• Decay by electron capture.
E .... (keV) 18 156 168 1700 35 27" 360 610
Relative abundance (%) 100 100 100 100 7 -80 87
Scintillation detector Liquid Liquid Liquid NaI, liquid NaI, liquid
294
G E N E R A L METHODS FOR EVALUATING HORMONE EF F EC TS
[21]
tritium (~H) with its very low fl energy is not one of the isotopes, and if samples are counted at "infinite thinness"--without significant selfabsorption. Indeed, until the advent of scintillation counters about twenty years ago, double-isotope experimental designs were rarely attempted. Scintillation Counters
Birks 1 has provided a thorough and basic review of the technology involved in "gamma" counters where the detector is usually an activated N a I crystal, which responds to as, ,/s, and K-X-rays and of liquid scintillation counters in which an aromatic phosphor in solution serves as the scintillator. These respond to fls as well as as, ~s, and K-X-rays. In both types of counter, the photoelectron spectra are proportionate to the energy spectra of the photons emitted from radioisotopic decay. " G a m m a " Counters
There is only slight degradation in the resolution of spectra in N a I crystal counters, so that isotope pairs with energies above the crystal's scintillation threshold can be easily separated for counting, with the proviso that overlap of the two spectra be ascertained with standards of the same geometry. Regoeczi and Wehber have recently shown that spillover of 131I into the ~2'~I spectrum varies with the well counter used and the 131I count rate. 2 For example, optimum channels in one of our automatic well counters yield an 125I efficiency of 37.6% and an 1'~I efficiency of 38.8%. The efficiencies depend not only on pulse height analyzer settings, but also upon the shielding and geometry of the crystal and of the counting chamber.) While the spillover of 125I into the ~3lI channel is inconsequential: 0.28%, the ~3~I contribution to the 1'-'5I channel is significant: 9.17%. Therefore 1~I cpm = A - - (0.0917 X B) if A represents the cpm in the ~2"~I channel and B represents cpm in the 1'~I channel. Although "gamma" counting does not involve corrections for complex quenching phenomena as does liquid scintillation counting, self-absorption of emitted energy may be quite significant and may vary with the isotope being counted. The examples of ~-~I and 1~I counting given in Table II should serve as a warning that standards for 7 counting should be as similar in geometry to the unknowns as possible. 1j. B. Birks, "The Theory and Practice of Scintillation Counting." Macmillan, New York, 1964. E. Regoeczi and C. Webber, I. Nucl. Biol. Med. 16, 10 (1972).
[21]
DESIGN OF DOUBLE LABEL E X P E R I M E N T S
295
TABLE II EFFECTS OF SAMPLE GEOMETRY ON RELATIVE DETECTION EFFICIENCY a
Isotope 125I 131I i~sI i~II 14C aH
Aqueous solution
Whatman No. 1 filter paper
Cellulose nitrate filter
A. "Gamma" NaI crystal scintillation counter 1.00 0.92 1.11 1.00 0.85 0.86 B. Liquid scintillation counter 1.00 0.65 1. O0 0.96 1.00 0.71 1.00 0.09
0.82 0.98 0.91 0.42
The counts per minute in a 0.1-ml aqueous solution of [~25I]NaI, [l~lI]NaI, [14C]adenine, or cyclic[SH] AMP were taken as 1.00 and compared to replicates air dried onto discs 20 mm in diameter. Gamma counting was carried out in a Nuclear Chicago automatic gamma system with a 2-inch NaI crystal using optimum channels for each isotope. The samples in 10 X 75 mm Pyrex test tubes were inserted into cellulose nitrate counting tubes. Liquid scintillation counting was with a Beckman LS-150 system with a wide channel encompassing the total spectrum of both isotopes. The aqueous samples were counted in toluene 7.0 g/liter PPO (2,5-diphenyloxazole), 10 % Biosolv BBS-3 (Beckman) solubilizer. The samples dried onto the filters were counted in toluene-PPO without Biosolv. Liquid Scintillation C o u n t i n g In most double-label experiments, two counting channels are used and the photoelectron spectra are only partially separated. In liquid scintillation counting there is too great a loss of detection efficiency if each isotope is eliminated entirely from the other channel: the photoelectron spectra of the fluorescence emitted from the organic phosphors are too broad. Indeed, if the photon energies are not widely separated, it is statistically impractical to eliminate the spillover of either isotope from the other channel. A decrease in detection efficiency can be produced by impurity or chemical quenching which interferes with the excitation of the scintillation phosphor by radioactive photons or by "color" quenchers which absorb the emitted fluorescence and thus interfere with the transmission of light to the photomultiplier tubes. The extent of quenching in a homogeneous solution of sample and phosphor in solvent will cause pulse height shifts of fluorescence spectra to lower photoelectron energies as an inverse function of the energy of the emitted photons. Quenching in samples will thus affect the detection efficiencies of two isotopes variably, the spectral
296
GENERAL
METHODS
FOR
EVALUATING
HORMONE
EFFECTS
[21]
shift of the lower energy isotope being greater. There is then no single set of optimum instrument settings for specific isotope pairs. Methods of quench correction for homogeneous samples (samples in solution or in translucent emulsions of fine micelles) are reviewed thoroughly in recent literature and will therefore not be dwelt upon here. A reading of references cited in footnotes 3-7 should provide a thorough grounding in theory and practice. I have summarized the principal attributes of the three approaches in Table I I I . Correction of the overlap of radioactivities must be carried out before quench correction. The questions t h a t deserve attention in this review are: (a) H o w are the cpm of one isotope mathematically best separated from the cpm of another? (b) H o w are the optimum instrument settings determined for a specific isotope pair? (c) W h a t are the restrictions on sample preparation for statistically reliable double isotope counting? M a t h e m a t i c s o] Double-Isotope Counting. One approach to doubleisotope counting is to continue to use the discriminator settings for single isotopes. Since each channel then counts contributions from both isotopes, simultaneous equations are necessary for the calculation of the absolute radioactivity (dpm) of each sample: H : radioactivity of the isotope H of greater E,la~ in the sample (dpm) L: radioactivity L of lesser E ..... in the sample (dpm) h l : detection efficiency of H in channel 1 (determined by sample channels ratios or external standardization, and expressed as a fraction of 1) h2: detection efficiency of H in channel 2 ll: detection efficiency of L in channel 1 12: detection efficiency of L in channel 2 N I : net cpm (cpm - - background) in channel 1 N2: net cpm in channel 2 H = [N1 - N 2 ( l l / 1 2 ) ] / [ h l - h2(ll/12)]
(1)
L = [N2 - N l ( h 2 / h l ) ] / [ 1 2 - l l ( h 2 / h l ) ]
(2)
3j. B. Birks, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 283-292. Grune & Stratton, New York, 1970. 4C. T. Peng, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 283-292. Grune & Str~tton, New York, 1970. M. P. Neary, and A. L. Budd, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 273-282. Grune & Stratton, New York, 1970. P. D. Klein, and W. J. Eisler, in "Organic Scintillators and Liquid Scintillation Counting" (D. L. Horrocks and C. T. Peng, eds.), pp. 395-418. Academic Press, New York, 1971. 'F. E. L. Ten Haaf, in "Liquid Scintillation Counting" (M. A. Crook, P. Johnson, and B. Scales, eds.), Vol. II, pp. 39-48. Heyden, London, 1972.
[21]
DESIGN OF DOUBLE LABEL EXPERIMENTS
297
TABLE I I I METHODS OF QUENCH CORRECTION IN LIQUID SCINTILLATION COUNTING A. Internal Standardization ("Spiking") Advantages Most accurate of any method if addition of a known standard to the sample is careful and reproducible. Differences in the effects of impurity and color quenching (which may be important with 14C and more energetic isotopes) are obviated Disadvantages The samples must be homogeneous; the standard must be similar chemically to the sample solute if there is any question of an incomplete solution. Handling errors in addition of the standard will be large if the procedure is careless; ideally this procedure should be done in duplicate. Once a sample is "spiked," it cannot be recounted. The procedure is extremely time-consuming. In double-label experiments, it is a laborious and statistically uncertain approach B. Sample Channels Ratio Advantages Data may be obtained in two channels simultaneously; thus, counting the sample only once is sufficient. The sample itself is not altered. Accurate for moderate and high count rates of single isotopes which are quenched slightly to moderately. Independent of sample volume over a wide range. One series of quenched standards will correct for both sorts of quenching of 3H. Nonhomogeneous samples (in suspension, emulsion, on solid supports) may be quench corrected, as long as standard curves are derived from known samples of the same composition and geometry Disadvantages The procedure is inaccurate for highly quenched samples or for samples with low count rates because strong color quenching of 14C or more energetic isotopes is not adequately corrected. With two overlapping isotopes, corrections for spillover must be made first, with a significant additional statistical error C. External Standard Channels Ratios This procedure, involving exposure of the unknown sample to an external 7 source takes advantage of the secondary Compton electron generated within the sample and the resulting broad fluorescence spectrum. Modern instrumentation monitors and subtracts sample cpm from two separate channels and then ratios the Compton cpm; single channel ES is undesirable inasmuch as the dependence of cpm on sample volume is considerable Advantages Application to samples of low activity. Statistically the most suitable for doubleisotope quench correction Disadvantages Least accurate for highly quenched samples in which differences between the photoelectron spectra of sample and Compton electrons are magnified. Samples must be homogeneous. This technique is not applicable to samples in suspension, on solid supports, etc.
298
GENERAL METHODS FOR EVALUATING HOR3IONE EFFECTS
[21]
The advantage to this procedure is that H can be counted at maximum efficiency unless there is severe quenching, but a considerable accumulation of statistical error results from the repeated application of calculated efficiencies and from the relatively complicated algebra. Instead we sacrifice some H efficiency: counting the higher energy isotope in a channel from which L is for all practical purposes excluded. In this case: g = N1/hl
L = IN2 -
(3)
(N1/h2)]/12
(4)
The propagation of error is greatly decreased by such a simplification. In our hands, analysis of the variance occasioned by the use of Eqs. (1) and (2) results in unacceptably large cumulative errors, particularly when count rates for one or both isotopes are low and the overlap is large. There have been several suggestions in the recent literature that simultaneous equations may reliably differentiate two isotopes with considerable spectral overlap. Such advice is dangerous; if simultaneous equations are employed the exclusion of isotope L from channel 1 should still be maximal. Since the effect of quenching on photoelectron spectra tends to this exclusion, such a stipulation is reasonable. The "spillover fraction" (N1 X h2) of Eq. (4) which is a constant in gamma counting, will increase in liquid scintillation counting as the spectrum of isotope H is quenched and progressively more H cpm fall in channel 2. In Beckman scintillation counters with "automatic quench corrections (A.Q.C.)" this fraction can be held relatively constant, s-" but in others, a quench correction curve for this fraction must be obtained, just as it is obtained for the detection efficiencies of the two isotopes in their optimum channels. Occasional investigators have attempted to use simultaneous equations to solve for the activities of three isotopes in the same sample, using two or more channels. The statistical errors implicit in such procedures are without exception unacceptably large. It is sometimes possible to separate one overlapping isotope from another, however, by combining different counting methods. 131I may be separated from 14C or 3~S, and 125I from all, by counting the samples in a gamma counter and then by liquid scintillation alone. It is important that 131I or 12~I efficiency be determined in both counters so that an aecurate "spillover" curve of either iodine isotope into the 14C, 35S, or 3H channel can be calculated from standards. This approach applies Eqs. (3) and (4) to data from two instruments rather than from one. 8 C. H. Wang, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 305-312. Grune & Stratton, New York, 1970. 9 M. F. Grower, and E. D. Bransome, Jr., Anal. Biochern. 31, 159 (1969).
[21]
DESIGN OF DOUBLE LABEL EXPERIMENTS
299
Energetic fls may be also counted in solution in the absence of phosphors by virtue of the Cerenkov effect?° Of the isotopes in Table I, ~P and ~31I can be counted at reasonable efficiencies without any impurity quenching, but Cerenkov photoelectron spectra are so poorly resolved that double isotope counting is not practical. Instrument Settings. The method of selecting discriminator settings for the single-channel counting of a single isotope is straightforward. Since background cpm increase with channel width, the upper potentiometer setting should be decreased to the point where the count rate of a sample of the isotope begins to become significantly diminished. The lower discriminator should be set close to 0 and raised only if there is a major contribution of instrument "noise" to background in the low energy range, a problem not significant in instruments of recent manufacture. In double-label counting there is an obvious conflict between the desires for minimum spillover and maximum efficiency. A general approach to the most effective compromise in any particular situation would obviously be of value. Klein and Eisler H have proposed a performance criterion, P. In the terms of Eqs. (1)-(4) P = hl ×12 X S
(5)
with hl and 12 the detection efficiencies of the two isotopes in their respective channels and S a function of the count ratios between the two channels for each isotope. Comparing a series of settings, however, may be difficult since, as is usually the case, one setting may be superior regarding only one or two of the three factors. Davies and Deterding'-' have introduced a less biased but more complex algebraic approach to find optimum settings, using a weighted sum of the activities of the two isotopes more or less independent of the contribution of background cpm, which I consider a bit too complex to review here. Graphical approaches which are perhaps a bit easier to comprehend are represented in Figs. 1 and 2.13 The procedure for choosing settings for channels for double-isotope counting in an LS counter with logarithmic or pseudologarithmic amplification is illustrated in Fig. 1. The most efficient channel for each isotope is determined separately as outlined above, and the effect of progressively lowering the upper discriminator in 20-division steps is plotted as the lo R. P. Parker, and R. H. Elrick, in " T h e Current Status of Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 110-122. Grune & Statton, New York, 1970. 11p. D. Klein and W. J. Eisler, A~al. Chem. 38, 1453 (1968). 12p. T. Davies and J. H. Deterding, D~t. J. Appl. Rad. Isotopes 23, 293 (1972). laE. D. Bransome, Jr. and S. E. Sharpe, III, Anal. Bioehem. 49, 343 (1972).
300 j
hi Z Z
GENERAL METHODS FOR EVALUATING HORMONE EFFECTS I00
C.;,
80
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i!iii::iiiiil::ililiii::ii::ii:j:~:,
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[21]
20 "
11 I00
200
300
400
500
600
700
800
900
I000
POTENTIOMETER DIVISIONS SETTING AT UPPER DISCRIMINATOR
Fie. 1. Method for selecting channels for double-isotope counting in counting systems with logarithmic or pseudologarithmic amplification. The cpm in cumulative 2% (20 potentiometer divisions) narrow channels are expressed as percentage of total cpm in the most efficient wide channel. The lowest discriminator setting reaching 100% on the ordinate is the upper end-point of the isotope energy spectrum in the specific counter being examined. From E. D. Bransome, Jr. and S. E. Sharpe, III, Anal. Biochem. 49, 343 (1972).
percent of total cpm in the full channel. The H channel chosen should be at the L end point so that there is no spillover of L into the channel used for H. With our Beckman LS-150 (Fig. 1), we found the ~31I channel to be between 260 and 660 discriminator divisions. The upper discriminator setting for the L channel can be decided by identifying the region of maximum difference between the higher and lower energy isotope plots. The shaded area in Fig. 1 indicates the area of proper choice. We chose the 0-260 channel for maximum 125I. A 0 - 1 4 0 channel would have been a better selection if the 131I activity tended to be considerably in excess of that of 1~5I, or if the unknown samples to be counted were considerably quenched so that the 1:5I photoelectron energy spectrum was significantly shifted to the lower end. For "linear" LS counters, the additional requirement of gain adjustment is the most important consideration involved in selecting channels for single- or double-isotope counting. It is not possible to comprehend the complete spectra of more than one isotope by varying the discriminator settings, unless the gain settings are significantly different. The effect of grain on counting efficiency in a wide channel should therefore be systematically examined. To minimize the effect of quenching, the maximum gain for a specific level of efficiency should be chosen so that as much as possible of the spectrum fills the window.
[21]
DESIGN OF DOUBLE LABEL EXPERIMENTS
301
bOO0%ds
Z
W
t J J
I00%.
i l d
f
i f J J i i d ,S 10%-
i J jJ S I
i
S S
S
i
16% iob~o 131I EFFICIENCY
ioOO~o
Fro. 2. Engberg plot of 125I and '31I in a Packard Tri-Carb 3375 LS counter. The efficiencies were determined in a fixed channel at various gain settings and plotted against each other (solid line). The best choices of isotope ratio can be identified by determining the greatest distance of the plotted curve from a 45 ° line drawn from the base (the dashed line). The best 1~1I channel is obvious: the gain with maximum efficiency and no 12~I spillover. For 125I, the best solution is a compromise between low ~31I spillover into the 1-'5I channel, and high 1~I efficiency. Maximal 1~I efficiency is accompanied by more than 20% spillover. If there is a substantial excess of ~1I dpm over 12~I dpm, a lower 1~I efficiency (at higher gain) should be offset by a further decrease in ~ I spillover. From E. D. Bransome, Jr. and S. E. Sharpe, III, Anal. Biochem. 49, 343 (1972). I f the absolute r a d i o a c t i v i t y of the i s o t o p e ( s ) being counted in a s a m p l e is not k n o w n , a comparison of cpm at each gain is sufficient, but there is an a d v a n t a g e to k n o w i n g counting efficiencies. K o b a y a s h i and M a u d s l e y 14 h a v e shown t h a t log-log plots ("Engberg plots") of isotope efficiencies in a fixed w i n d o w can be used to determine the ability of a linear LS counter to separate an isotope pair. Figure 2 shows our results counting 131I and 125I with a P a c k a r d 3375. B e c a u s e the effect of quenching on the lower energy isotope increases the isotope separation, using o n l y l e a s t - q u e n c h e d isotope standards to set up counting channels is a sufficiently rigorous procedure. 14y . Kobayashi and D. V. Maudsley, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, Jr., ed.), pp. 76-85. Grune & Stratton, New York, 1970.
302
GENERAL METHODS FOR EVALUATING HORMONE EFFECTS
[22]
Sample Preparation. Most of the important considerations of sample preparation for liquid scintillation counting can be conveniently reviewed in references cited in footnotes 15 and 16. It is worth reiterating that for calculations of the radioactivity of an isotope pair to be reliable, both isotopes must be uniformly distributed in the sample. Translucent emulsions with microscopic micelles may be counted as well as true solutions if three cautions are observed: (a) If one of the isotope pair is 3H or 1~5I, it must be in the same phase as the other isotope. This might seem to be a trivial problem since emulsion counting is of aqueous samples, but it is not; there may be differential extraction of the labeled solutes into the organic solvent. (b) Absorption (of the lower energy isotope in particular) onto the vial surface may greatly alter detection efficiency.17 (c) If the E .... of an isotope is equivalent to or greater than 14C, and a commercial solubilizer of aqueous samples has been included in the sample, the surfactant itself may fluoresce in response to radioactivity. This renders quench correction curves derived from sealed commercial standards invalid2 s All these proscriptions may be summarized in the cardinal, and frequently neglected, general rule for scintillation counting: standards should be of the same geometry as the unknown samples. 15ff. C. Turner, "Sample Preparation for Liquid Scintillation Counting," Review 6. The Radiochemical Centre, Amersham, England 1967. Reprinted in "Handbook of Radioactive Nuclides" (Y. Wang, ed.) pp. 256-273. Chem. Rubber Publ. Co., Cleveland, Ohio, 1969. '6"The Current Status of Liquid Scintillation Counting" (E. D. Bransome, ed.), Chapters 16-27. Grune & Stratton, New York, 1970. 17G. ft. Litt and H. Carter, in "The Current Status of Liquid Scintillation Counting" (E. D. Bransome, ed.), pp. 156-163. Grune & Stratton, New York, 1970. ,8 S. E. Sharpe, I I I and E. D. Bransome, Jr., Anal. Biochem. 56, 313 (1973).
[22] U s e o f A n t i b o d i e s t o N u c l e o s i d e s a n d N u c l e o t i d e s in S t u d i e s o f N u c l e i c A c i d s in Cells
By B. F.
ERLANGER, W . Y. KLEIN, JR., V. G. DEV, R. R. SCHRECK,
and 0. J. MILLER The preparation of purine- and pyrimidine-protein conjugates and their use in eliciting base-specific antibodies that react with nucleic acids have been described in this series, Volume 12B [173]. Their reaction with nucleic acids requires that the purine or pyrimidine bases be unpaired, i.e., that the nucleic acid be denatured or, at least, have single-stranded regions. It follows, therefore, that demonstrable reaction with these antibodies is evidence for "single-stranded areas." Fluorescein-tagged
