Instrumentation 3
2
1
10
20
30
40
50
60
70
80
90
100
Relative Isotope Abundance (Atom '01
Figure 1. Percent accuracy and precision of isotope abundance measurements as functions of relative abundance on gases introduced from reservoir
Direct Analysis of Stable Isotopes with a Quadrupole Mass Spectrometer R. M. Caprioli Department of Chemistry Purdue University West Lafayette, Ind. 47907 W. F . Fies and M. S. Story Finnigan Instrument Gorp. 595 N. Pastoria Avenue Sunnyvale, Calif. 94086
Isotope ratios can be determined w i t h high precision by quadrupole mass spectrometry e v e n under the dynamic conditions imposed by com bined gas chromatography-mass spectrometry. Applications abound especially in chemistry, biology, and medicine
Interest in the use of stable isotopes has sharply increased in recent years in all fields of chemistry, biology, and medicine as a combined result of a decrease in the cost of stable isotopes, advances in the design and performance of magnetic resonance and mass spectrometric instrumentation, and the general concern over the hazards of radioactive isotopes. Of particular utility in isotope methodology is the emergence of mass spectrometric techniques designed to simultaneously determine the abundance of a particular isotope and also its location within the molecule. This direct analysis thus provides much structural information and has considerable advantage over other isotope techniques which require complex degradative procedures to locate the isotopic atom. Stable isotopes have been used in a wide variety of applications, most of which involve their use as tracers either to follow the fate of a particular atom in a reaction series or an entire molecule in a complex system. For example, tracing particular atoms of a molecule is vital to the elucidation of the origin of certain atoms of cellular metabolites in biosynthetic stud-
ies ( I , 2 ) or in the study of reaction mechanisms where the fate of one or more atoms is diagnostic of a particular mechanism (3, 4 ) . Specific isotopic labeling is also used when the fate of the entire molecule is of interest, such as in studies of the metabolism of drugs or other compounds in living cells ( 5 )or in isotope dilution experiments in which quantitative analyses are sought. In view of the increasing use of stable isotopes, it is becoming of critical importance to develop mass spectrometric techniques which attain better accuracy in the measurement of isotope abundances in complex molecules. In addition, the use of the technique of mass fragmentography in which samples containing mixtures are analyzed by continuous ion monitoring also requires accurate ion intensity measurements if quantitative data are to be obtained. This is of significant value in the analysis of drugs, drug metabolites, and other compounds a t subnanogram levels (6). Direct Analysis of Stable Isotopes
Direct analysis of stable isotopes involves measurement of ion intensi-
A N A L Y T I C A L C H E M I S T R Y , VOL. 46. NO. 4 , A P R I L 1974
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ties of the several isotopic species of a molecular or fragment ion formed in a mass spectrometer. In addition, if structural information is required, a study of fragmentation reactions through the use of normal and specifically labeled compounds may also be necessary. The alternative method for the mass spectrometric analysis of stable isotopes is indirect and involves the combustion of the sample to a gas, usually Hz for deuterium analysis, Nz for l5N analysis, and COz for 13C and 1 8 0 analyses, followed by measurement of the isotope ratios of these gases (7). However, this method has the disadvantages of providing no structural information; requiring relatively large amounts of sample, usually a t least 1mg; and poor accuracy as a result of the simultaneous combustion of contaminants or incomplete combustion, even though the mass spectrometric ratio measurement may have a precision as high as &0.001%. Although direct analysis of stable isotopes is the method of choice in many cases, it has not become a routine tool because of the relatively poor precision obtained in isotope ratio determinations by use of magnetic mass spectrometers. To be of general utility, methods for direct analysis must be able to achieve a precision of approximately &0.1% or better with microgram or nanogram quantities of sample injected into the GC inlet of a mass spectrometer. Many biomedical experiments involve the isolation of small amounts of samples containing low isotope abundances. In many cases, even high initial isotope concentrations give low abundance products owing to the enormous dilution of the isotopic compound by the system. In addition, better precision in measurement would also permit the use of isotope methodology in low enrichment experiments where, previously, the high cost of high enrichments was prohibitive. To achieve high precision in the measurement of the intensities of two or more ions, it is desirable to measure these ions simultaneously in the manner of double collector techniques used with isotope ratio mass spectrometers. However, the fixed focus of magnetic instruments and use of a Faraday cup collector, which are essential to the high precision achieved with this method, do not lend themselves to ratio measurements on high mass and low abundance ions. The potential solution to these problems lies in the utilization or repetitive scanning techniques. Thus, Hites and Biemann (8) used continuous rapid scanning over a given mass range to measure ion intensities. Although this was an improvement over data ob454A
tained from individual mass spectra, scanning a portion of the mass spectrum in this manner results in poor ion statistics since a great deal of time is spent between peaks where ions are not collected. In another approach, Sweeley et al. (9, I O ) , Klein et al. ( I I ) , and, more recently, Holmes et al. (12)and Watson et al. (13)used an accelerating voltage alternating (AVA) device to continuously switch the accelerating voltage in a cyclical manner so as to successively focus a series of ions a t the collector. The precision of isotope ratio measurements achieved with this method was approximately &I%. Limitations of the AVA technique are detuning of the ion source when the accelerating voltage is changed, relatively slow switching rates demanded by the high voltages involved, and the requirement that the mass difference in ions to be compared may not be greater than about 40%. Advantages of Quadrupoles Quadrupole mass analyzers present some attractive features in the pursuit of higher precision isotope ratio measurements. First, the electrostatic voltages used to produce mass dispersion can be switched rapidly and measured accurately. Thus, in a high switching rate mode, each ion of an isotope series would be collected for a time on the order of milliseconds with continuous cycling over the series of ions to be measured. Such a system would approach the ideal of simultaneous collection of ions used with the double collector method. Thus, with rapid switching, only those instabilities or pressure changes comparable to the switching rate will affect the abundance measurements. Since the dead time or settling time between masses is small ( - 1 msec), almost all the time the sample is in the instrument is spent collecting the ions of interest. Thus, compounds emerging from the GC having a duration of several seconds can also be analyzed. Second, ions in a particular series may be collected for different amounts of time depending on their relative abundances, maximizing ion statistics. Third, any ions in the spectrum, no matter their mass difference, may be measured in a particular analysis and need not lie within a given percentage of the mass range. During the past five years, the improvement of quadrupole mass spectrometer design and performance has virtually eliminated difficulties generally associated with these instruments, such as lack of high mass sensitivity and peak-tailing, and has provided capabilities which are comparable to magnetic deflection instruments in this regard. Quadrupole mass spec-
ANALYTICAL CHEMISTRY, VOL. 46, N O . 4 , APRIL 1974
trometers, already in the forefront of GC/MS methodology, further hold the potential of becoming a primary instrument for the direct analysis of stable isotopes. In the work described here, isotope abundances were determined on a variety of samples with a quadrupole mass spectrometer. These include some of the inert gases and 180-labeled sugars. The inert gases are ideal samples for testing both precision and accuracy of isotope abundance measurements because their abundances are known exactly and because there is a single isotopic species per mass, unlike organic compounds where there may be mixtures of carbon, hydrogen, nitrogen, and oxygen isotopes a t a particular mass. In addition, the range in the relative abundances is large, being nearly 16OO:lfor m l e 40:mle 38 for argon and 1:1for certain isotopes of krypton and xenon. Thus, in the experiments which follow, Ar, Kr, and Xe were employed as standards. Their isotope abundances were measured by use of a data system to analyze samples introduced both from a reservoir inlet in which the pressure is stable and from the GC inlet in which the pressure is constantly varying. The measurements obtained from the data system were also compared with those obtained with the programmable multiple ion monitor (PROMIM) coupled to digital integrators. A second series of experiments was performed involving measurement of isotope ratios in fragment ions of natural abundance and 180-enrichedpentaacetylglucose samples introduced into the mass spectrometer via the GC inlet. This experiment was also performed with the multiple ion monitor-integrator system, and the results were compared with those obtained from the data system. The precision and accuracy of the measurement techniques are given together with a discussion of critical parameters involved in such measurements. Experimental
The inert gases (analyzed grade) were obtained from Mathieson Gas Products. (2-180)and 6-lS0)pentaacetylglucose were prepared and analyzed as previously described (12). A Finnigan Model 3100 quadrupole mass spectrometer equipped with a Model 9500 gas chromatograph and a Model 6000 data system was used for the determination of isotope abundances. For ion detection, a 14-stage beryllium-copper electron multiplier was employed. The mass spectrometer was equipped with a 1-liter reservoir inlet system for the introduction of gases. For GC studies, a 3-m x 2-mm
(i.d.1 OV-1 column was used. A mass defect adjust device was installed in the mass calibrate circuit so that after calibration with perfluorotributylamine, the mass set point could then be moved the proper fraction of a mass unit, depending on the mass defect of the ion to be measured, to monitor the top of the mass peak. That this indeed occurred was verified by using the “diagnostic” mode of the data system which allows the operator to observe on the oscilloscope display the peaks of interest and the point on these peaks a t which the instrument is calibrated. Ion intensities for the isotope abundance determinations were obtained by using the mass fragmentography program of the data system in which, for a given mass, ions are collected through a series of sampling times, 1, 2, 4, 8, 16, 32, and 64 msec or until the analog-digital convertor saturates. At this point, the computer will store the total signal intensity accumulated a t the time prior to saturation and then shift to the next ion in the isotope series and repeat those operations. Thus, the time spent collecting ions of a given mass varied from 1to 64 msec, depending upon the intensity of the ion current. After the last isotope in a series was measured, the system immediately recycled to the first, providing continuous monitoring throughout the analysis. Isotope ratios were determined by using the area under the peaks produced by the mass chromatograms, i.e., the curve produced from a plot of ion intensity vs. time for a given isotope. A total of four ions could be monitored a t one time. For isotope series containing more than four ions, one ion was arbitrarily chosen as unity, and the others were calculated as a ratio of this. The abundance measurement of the ion normalized on was repeated in each set of determinations until all isotopic species were measured. The oscilloscope display of the data system allows the mass chromatogram to be observed as the data are being acquired. When this is complete, the operator can then choose the two points on the mass chromatogram through which a base line should be drawn. The two points between which the peak area is to be calculated are then chosen, and finally the peak area is obtained in arbitrary units or as a voltage, with the portion of the background under the peak subtracted. Other ions in the isotope series are then similarly measured and their relative abundances calculated. The precision of the measurements presented in the tables which follow is given in terms of the mean deviation as a percent of the average value 456A
calculated from four to six independent determinations of each isotopic species. The percent accuracy of the measurements is given as the percentage by which the average abundance differs from its true value. In the experiments where the programmable multiple ion monitor (PROMIM) was used, each isotopic species was monitored on a separate channel of the unit, and the ion current measured by individual digital integrators (Autolab Model 6300). The ion monitor was modified to sweep over the top of each peak covering a range of approximately 0.25 amu, rather than remain stationary. Sampling times of 1,10, and 100 msec could be chosen for each sweep. As before, the instrument switched from peak to peak continuously throughout the analysis.
Results Reservoir Samples. The relative abundances measured for the inert gases are given in Table I. The gases were admitted from a 1-liter reservoir a t room temperature through a molecular leak to give an indicated ion source pressure of approximately 5 x 10-7 torr. Measurements were obtained with the data system in the mass fragmentography mode, as described earlier, with a total sampling time of 5 min. Since a maximum of four ions could be monitored simultaneously, for krypton and xenon, isotope abundances were determined as ratios of mle 82 and 129, respective-
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 4 , APRIL 1974
ly, and then relative abundances calculated. The precision and accuracy attained as a function of isotope abundance are shown in Figure 1. GC Samples. T o make a direct comparison of the precision and accuracy possible between the reservoir gas sample where the pressure is not changing and a more dynamic case of GC samples where sample pressures are constantly changing, xenon and krypton were introduced via the GC inlet a t room temperature by using a gas-tight syringe. The results are given in Table 11. As a typical example, Figure 2 shows the mass chromatogram of xenon a t m / e 124, the lightest and least abundant isotope, taken on continuous monitoring with the injection of four different samples. Each GC peak was of approximately 8-sec duration. The abundances of each of the four peaks shown in Figure 2 were, from left to right, 0.098%, 0.093%, 0.095%, and 0.095%, with the true value being 0.096%. The greater precision of the last two reflects the effect of better ion statistics. Comparison of the data given in Table I1 for GC analyses to that in Table I for reservoir analyses shows comparable accuracy and precision. Thus. it can be concluded that with the rapid peak-switching system used here, changes in sample pressure encountered with GC samples are sufficiently slow compared to the switching time so as to have no significant effect on abundance measurements. Isotope abundances of fragment
ions of organic molecules were also measured. In the mass spectrum of pentaacetylglucose, the ion of m l e 242 is derived from the molecular ion by the successive loss of acetic anhydride and formic acid ( 1 3 ) .Thus, the CH.04c
OAc
OAc m / e 288
nt e 390
J L
ion a t m / e 242 contains the C-2, C-4, and C-6 oxygen atoms of the original glucose molecule. Samples of (2-180) and (6-l80) pentaacetylglucose, as well as unenriched pentaacetylglucose, were dissolved in methanol to a concentration of approximately 1 Fg/pl and were injected into the GC inlet system a t a column temperature of 220°C. The ion intensities a t m l e 242 and 244 were measured, and the isotope abundances of mle 244 are given in Table 111. The mass chromatogram for unenriched pentaacetylglucose is given in Figure 3. The precision obtained with these samples was the same as that obtained from the analyses ofthe inert gases. A final set of experiments was performed in which the multiple ion monitor-integrator system was used to measure isotope abundance of GC samples. Since each channel of the
Table 11. Isotope Abundance Measurements on Some Inert Gases as GC Samples Theoretical measd abundance,
Measd re1 abundance,
R
Accuracy,
Precision,
R
%
Sample
mle
%
Krypton
78 80 82 83 84 86
0.35 2.27 11.56 11.55 56.90 17.37
0.34 0.004 2.24 = 0.003 I, 11.45 11.59 i 0.034 56.89 = 0.09 17.48 I 0.02
-2.9 -1.3 -1.0 +0.3 -0.02 +0.6
e1.2 ~0.14
124 126 128 129 130 131 132 134 136
0.096 0.090 1.92 26.44 4.08 21.18 26.89 10.44 8.87
0.095 = 0.001 0.090 & 0.001 1.98 0.016 26.49 4.10 + 0.025 21.05 = 0.04 26.81 I 0.03 10.49 = 0.08 8.89 = 0.06
-0.1 0.0 +3.0 +0.2 +0.5 -0.6 -0.3 +0.4 +0.2
X1.1 *1.1
Xenon
f1
6
=0.29 i0.16 xo.11
*0.8 I,
k0.61 10.19 10.11 &0.76
~0.67
“ H a n d b o o k of C h e m i s t r y a n d Physlcs,” 51st ed., Chemical Rubber Co., 1971. normalized o n . n
Figure 2. Mass chromatogram of xenon at m / e 1 2 4 taken on continuous monitoring of four separate sample injections. Each scan number in plot represents 1 sec 458A
*
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9
Ion
Table 111. Isotope Abundances of Fragment Ion m / e 244 of Labeled and Unlabeled Pentaacetylglucoses Introduced Via GC Inlet Measd re1 abundance, Sample
Unlabeled .?-‘SO 6-1’0
%
3.02 i 0.04 36.27 i 0.03 49.80 i 0.05
Precision,
%
+1.3 5~0.08
=tO.lO
ion monitor was connected to a separate digital integrator, it was necessary to calibrate the integrators to obtain a valid comparison between this system and that used earlier. The calibration was performed by tuning the two channels of the ion monitor t o the same mass by using a 100-msec sampling time and comparing the integrated peak areas from each channel for different amounts of sample. Figure 4 shows this calibration curve plotted as integrator units vs. the ratio of the areas. When the area of the peaks was greater than 4 x 105 units, the ratio of the areas from the two channels was constant. In the measurements which follow, sensitivity was adjusted so that the ratios fell in the linear portion of this curve. In a typical analysis, one or two sample injections are required to set the mass of each channel a t the top of the proper peak and to set the sensitivity range of each channel if the approximate abundances are not known. Subsequent sample injections can then be used for the actual isotopic abundance measurements. The isotope abundances of three samples admitted via the GC were measured in this way-krypton and the two 180-enriched glucoses-and the results are given in Table IV. The precision achieved was again similar to that obtained in the earlier experiments. Discussion One of the critical factors in determining isotope abundances with a mass spectrometer is peak shape. Thus, whether dealing with a magnetic or quadrupole instrument, it is important to adjust the ion source controls to give rounded tops-the closer to a flat top, the better the precision which may be obtained. However, since this type of “tuning” decreases the sensitivity of the instrument, a compromise must be made to favor one or the other depending upon the particular application. In the work reported here using a quadrupole, ion source tuning conditions for the attainment of a precision of
0.1-0.2% resulted in a concomitant loss of a factor of only approximately 2 in sensitivity. Since these instruments can generally obtain mass spectra from as little as 1 x gram of sample, this loss of sensitivity is not of major significance. Resolution also plays a significant role in isotope measurements. At maximum resolution, the peaks become extremely sharp, making it more difficult for the mass set to remain on top, since any instabilities will cause it to shift to the side of the peak. On the other hand, a t low resolution the tail of one peak will add to the intensity of adjacent peaks, giving poor accuracy. Therefore, resolution was chosen to no more than a 1-2% valley between adjacent peaks, providing a good tradeoff between resolution and accuracy. Inspection of Figure 1 shows that for d a t a obtained for gases sampled from the reservoir, the average precision obtained was *0.1510 in the abundance range 10-100 atom %, k0.470in the range 1-10 atom %, and *0.9% in the range below 1 atom 70. The rather sharp falloff in precision when the isotope abundance being measured is below 1-2 atom 70is primarily the result of poorer ion statistics. Also from Figure 1, the average accuracy obtained for the reservoir gases was 0.4% of the true value in the abundance range 10-100 atom 70, 1.4%in the range 1-10 atom %, and 3.6% below 1 atom 70.The decrease in accuracy a t low abundances is the result of the combination of several factors. First, a t low resolution, a small amount of cross-talk takes place between peaks and has its greatest effect when the ratio of adjacent peaks is high. Second, any background peaks present will make a larger percent contribution to the lower abundance isotopes, as also will any systematic errors in the measurements.
m/e 244
m/e 242
1
100 Scan Number
150
Figure 3. Mass chromatogram of m / e 242 and 244 of unenriched pentaacetylglucose. Each scan number in plot represents 1 sec
Figure 4. Calibration curve for multiple ion monitor-integrator system with m / e 242 of pentaacetylglucose introduced via GC inlet as a standard. Curve plots arbitrary integrator area units for each mass chromatogram vs. ratio of these area units for two channels used
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A comparison of Table I (reservoir inlet) and Table I1 (GC inlet) shows that although the precision of the measurements is about the same in both cases, the accuracy of the low abundance measurements is better for samples introduced via the GC inlet. This is not surprising since the data system allows a base line to be drawn for the mass chromatogram peak and background subtracted from the area of this peak to a close approximation. This could not be done for the reservoir samples, and any residual background was included in the measurements. Thus, these data show that rapid switching techniques with a quadrupole mass spectrometer can be used to analyze GC peaks for isotope abundance without sacrificing accuracy. Perhaps a more rigorous test of the technique used here is the isotope abundance analysis of the fragment ions of normal and IsO-labeled pentaacetylglucoses introduced via the GC inlet. The data in Table I11 again show excellent precision, in agreement with that obtained with the inert gases. This, of course, provides the capability of simultaneously obtaining structural information and higher precision isotope ratio analyses, giving both the position of the isotope within the molecule and its abundance a t that position. The experiments with the multiple ion monitor-integrator system show that such a system may also be employed for high-precision isotope measurements, as shown in Table IV. This system has the main advantage of being less expensive than the data system. On the other hand, with the data system it is easier to focus the masses t o be monitored. This system is also more flexible in that it has the ability to adjust for maximum integration time without prior knowledge of the isotope abundance, the capability of directly determining the ratio of areas by an “area ratio” function, and the capability of allowing the operator to visually choose the two points through which the base line is drawn. The latter is of major importance and is illustrated by the comparison of the isotope abundances of the I*Oenriched glucose samples from the data system and ion monitor system. In both cases, the abundances obtained with the ion monitor are lower by 0.2 to 0.3 atom 70I 8 0 than those with the data system. This was due to the presence of an impurity in the sample, giving a small peak at m l e 242 on the leading edge of the sample peak, and resulting in a base line change, but there was no corresponding peak a t m l e 244. Thus, the digital integrators on the ion monitor added this shoulder into the peak
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area a t m l e 242, giving a lower abundance for m l e 244. The data system, on the other hand, allows visual inspec, tion of the peak and provides the ability to go back after all the data are taken and choose the base line to a closer approximation. Thus, in the above case for the data system, this shoulder was not added to the peak area for m / e 242. One further point dealing with isotope abundance measurements with a quadrupole mass spectrometer is worth noting and concerns mass discrimination. Of the several isotopic series studied, the accuracy attained with the low mass species was the same as that for the high mass species, within the abundance limits discussed above. Thus, no mass discrimination was observed, e.g., in Tables I and 11, the relative accuracy obtained for the xenon isotopes as one proceeds from low to high mass shows no trend as a function of mass within these 12 mass units. In conclusion, the work presented here demonstrates the capability of a quadrupole mass spectrometer for use as a high-precision instrument for the determination of isotope ratios, even under the dynamic conditions imposed by combined gas chromatography-mass spectrometry.
Acknowledgment The authors thank Autolab Inc., for the use of two of their Model 6300 digital integrators.
References (1) G. Waller, R. Ryhage, and S. Meyerson, Anal. Biochem., 16,277 (1966). (2) R. M. Caprioli and D. Rittenberg, Biochemistv, 8,3375 (1969). (3) M. Cohn, Biochim. Biophys. Acta, 37, 344 ( 1960j . (41 H . F . Fisher. E. E . Conn. B. Vennesland, and F. H . Westheimer, J . Bioi. Chem , 202,687 (1952). (5) H. Eriksson, J. A. Gustafsson, and J . Sjovall, Eur. J . Biochem., 9,550 (1969). (6) C. G. Hammay, B. Holinstedt, and R. Ryhage, Anal. Biochern., 25, 532 (1968). ( 7 ) R. M. Caprioli, in “Biochemical Applications of Mass Spectrometry,” G. Waller, Ed., p 735. Wiley-Interscience, New York, N.Y., 1972. (8) R. A. Hites and K. Biemann, Anal. Chem., 42,855 (1970). (9) C. C. Sweeley, W. H. Elliott, I. Fries, and R. Ryhage, ibid.,38, 1549 (1966). (10) J. F. Holland, C. C. Sweeley, R. E. Thrush, R. E . Teets, and M . A. Bieber, ibid.,45,308 (1973). (11) P. D. Klein, J. R. Haumann, and W. J . Eider, ibid.,44, 490 (1972). (12) W. F. Holmes, W. H . Holland, B. L. Shore, D. M . Bier. and W. R. Sherman. ibid.,45,2063 (1973). (13) J. T . Watson, D. R. Pelster. B. J . Sweetman, J . C. Frolich, and J . A.
Biochzm. Biophys. Acta, 297, R. M . C . thanks the American Cancer Society for partial support.
