Assay of Samples Doubly Labeled with Radioactive Hydrogen and Carbon \IAX W. BIGGS, DAVID KRITCHEVSKY, AND AIARTIU R. KIRK Donner Laboratory of Medical Physics and Radiation Laboratory, University of California, Berkeley 4 . Calif.
1
URING investigations of various aspects of cholesterol metabolism, a need arose for a simple method of assaying biological specimens doubly labeled with both tritium (HI) and carbon 14. The apparatus and procedure described belon- are applicable to problems requiring simultaneous information on both hydrogen and carbon turnover, and have satisfied the authors' needs. Both carbon 14 and tritiuni specific activities can be determined with a reawnahlc degrrr of accuracy n-ith a single samplc corntiustion, and the determinations are readily performed on samples providing approsima tel>150 mg. of water on combustion. Siri (4)in this laboratory has iiivest,igatcd various methods of assaying tritium activit>-, and l i p has found t.he reaction of lithium aluminum hydride with n-atcr suitable and convenirnt for the generation of hydrogrn from wat,er w n p l r s containing tritium. The authors are in~lchtpcito his experience and to his gencrour; ndvico during the developnient of the filling system for tritium :issay used in this investigation.
Fine f r i l l e d _glass
Figure 1.
vacuum souae
LLqy&? nilrogen- baJh
Figure 2.
Filling Y:striri for Ckrirrating ' r r i t i l ~ r ~ i - I r ~ d r oSamples g~ll I'REPAR iTION 01; S . i \ I P L E S
The labeled compounds usctl w r r rholesterol(random labeled biologicall) trrial), specific activity 6.67 X ld-3pc. prr mg., naphthoic acitl-carbosy1-C14, prepared by carbonation of the approprintc (;rignard reageri t n-ith isoto1)ic carbon diosidr (i?),specific activity 8.25,~. per mg., and tritium-labelcd cliolcsterol prepared hy eschange hetwrrri ciiolestc~rol and tritium osidc (3),specific activity 0.921pc. perm^. .In aliquot of an alcoholir solution of the alliiropriatr carhon-lahelcd conipountl ivas carefully transferred t o a large quartz comhustion vessel. and the solution KRS evaporated to dryness. After the material was dry, tritium-labeled cholesterol was added in t.he same way. Finally, a wighed amount of vacuum-dried cholesterol as added to the combustion vessel, this amount calculated to give sufficient n-ater for tritium assay (generally 150 mg.). The samples were varied to give a representation of carbon and tritium activities. Naphthoic acid was substituted for cholesterol after a few experiments in order to conserve hiologically prepared material and also to affonl samples with a higher carbon 14 activity. (:I'
I
k&)
lherrnocougle
filler,
n
'
To
Carbon dioxide absorber
1 /I
~
Coinbustion Train Used f o r I h u h l y Labeled Sample.; COMBUSTION OF SAWPLES
Sa111glt. so.
Nalilitliuic hcid-Carboxyl-Cl4 Activity I ctirity calcd., found, '.; deviarc./sampic ec. J s a m p l r vistion
1
0
2
1)
? 1 1;
a
0 in 11 12
' 3
.... ....
1) (i 1
n.Ri A;
0 3.1
1,29
1.20 1.27 3.19
7.n 1.li l ?
0.65
1.29 3.28
2.23 4.85
4.85
G.47 6.47 3 3 . 3 5 X 10-8 16.68 x 10-3 5 0 . 0 3 X 10-3
n
3.16
1.5
1.83 4.74 6.45 6.49
Cholesterol-C1 4 .7 3 1 . 2 3 X 10-3
0.4 2.3 0.3 0.3 = h2.7 6.4 8.2 0.2
18.01 x 10-2 4 6 . 9 5 x 10-2
C
=
==in
Activity calcd. pc./samplc 0.1,i.i 0.O i i
n.11n
n.iin
n.0;; 0.077 n. 038 0.038
n.om
Ciiolesterol-1%3 Activity found.
~~
% deviat ion :3 , [I 3 .0 ;
pc./sanipl~~
0.148 0.074 o, in;
I,
0 . Ion 0.076
Figure 1 is a schematic representation of the combustion train used. Trap .1 i.c submerged in an ice-salt bath to collect the tritium oxidewater sample and is designed to fit directly into- the filling Pystem for tritium assa!.. Quantitative d e c t i o n of the water formed (luring coinhnstion is n o t rcquircd.
0 4
n.ni6
I .R
1.3
TRITIti\l ASSAY
0,040
r, . 3 -5 3 7. I
Figurc 2 shows the filling Pysteni u w i for tritium assay. Trap d containing the tritium osidewater sample for assay was transferred to the evacuated filling system (stop~ o c k 1 closed). Tube B contained :< ml. of a saturated solution of lithium aluniinum hydride in anhydrous Carbitol. The water sample was frozen in liquid nitrogen, stopcock 1 was opened,
0.036 0.014;
0.0154 0 0
no m
n e
0.110
0 108
0.077 0 038
0.074 0.036
1 u .3 Y
...
0
-_____-
=
5.3 =k3 $9 .___
223
ANALYTICAL CHEMISTRY
224
and thelithium aluminum hydride solution was outgassed. When outgassing was complete, stopcock 1 was closed and the water sample allowed to melt. The lithium aluminum hydride was quickly added to the water sample (stopcock 2 closed; stopcock 1 open), and the evacuated system wm filled with the generated hydrogen-tritium mixture. The sample of hydrogen was then forced into the ionization chamber via a modified Toepler pump to nearly 1 atmosphere pressure. Finally, stopcock 3 vas opened and the contents of the ionization chamher were adjusted to a t mospheric pressure by the ,nercury levelsin the side a ~ m and pump chamber. The in the ionization chamber wBs determined using a vibrating reed eleotrometer. The drift ohtnined NBS corrected to standard temperature and pressure. ~h~ m ~ ~ and ~ u ~ ofy filling s,,stem and the of the ionization chambers showed no bothersome memory, and pumping for 30 minutes n-a8 nufficicnt to dear the sy8tem for the nest analysis. CARBON ASSAY
The carbon dioxide generated during the combustion vas collected in sodium hydroxide solution and precipitated as barium
oarbonate. The barium carbonate, after dryin N&S plated on aluminum disks by the method described by 8alvin et ai. ( 1 ) . The activity was determined using 8. Xueleometer windowlesstype counter. Several plates were prepared for each sample and the average of several determinations NBS used in calculating the final activity in each case. Greater accuracy, if desired, c m be obtained in the caw oi weak samples ifthe carbon 14 is nssayed as carbon dioxide in an ionisntion chamher instead of using harium earbonat,e plates. LlTER4TURE CITEII (I) Calvin. >I.. el 01.. "Isotovic Cnrhoo." New Pork. John Wiley &
Sons,1949.
(2)
be^. m.G.. Reid, J. c.. and Yanku,ioh, P. E., .~XAL.CHEM., 19,828 (1947).
(3) Rritehevaky, D.. Biggs. M. K., and Fmomim. Radiation Laboratory, Ptch. 644 (1950). (4) Siri. 17.. unpublished research. RECEITED
J U I ~14. i o i l .
work enonsored by
K. K.. Univ. Calif.
u. s. .itomie
~ n e r Comx ~
mission.
Simple Photometric Method for Use with RCA Type EMB Electron Microscope W. G . KIRCHGESSNER, Rausch & Lomb Optical Co., Rochester, R: P. S THE photographic process the supcess or failure of a print the tonal values in t.he original euhject. CouEequenbly, in electron microgrnphy, il correct CXDORure should hc such t h a t the lieht intensities from the unshndowed area of the suhject heiug photographed fall on of the D - log curve. T~ this the straightline
1depends upon the sat.isfactary rendering
~~
~
condition satisfactorily a photometer is needed, but hecause of the low intensit.\- values encountered in this txwe of uork.. ordinary photoelecdric meten are inadequate. I n view of this limitation, method for objt,aining erposure dab v~~ Bought and .ZR a 8implo ~ n devised, 9 advantages Over previously suggested designs (1-8).
".
The amplifier unit is a Kelch Densichron photoelectric control unit mounted on a bracket att.ached to the side of the microscope. Light rndiiLt,ionsfrom the fluorescent viewing screen in the microscope concentrated by ta-o aspheric condensing lenses (equiv-
nlont \ rill tho *athnrla nf d l ...~.." fnrnrr = 7.K _"mm _______, ". tho nhntndootrir r__l"l".-l"___ I_..
(Cetron CE-ZSV) which is attached to the lens mount. By this xrangement the photoelectric cell receives only light t h a t is reflected from the screen, and adequate needle deflection on the isobtained for thr low intensity values generally-
omnlovrrl .~ _... ~ .
~
.
~
~
Kith t,he unit attached ns desoribed, calibration m s made hy rorrel8,ting t,he relationship betxeen photographic densities. evposurc t,inles, and meter readings. Experiment.ally this n.a.8 done by csposing a series of test negatives for different unit,s of timc a t a standard light intensity as determined hy selection of some constant Densichron meter value. Control of this variable is maintained hy adjustment, of the condenser lens current on the microscope. With tho test negatives exposed to B series of known exposures and proceesed under identical and controlled rwnditionr, the responses of the photographic emulsions usetl \,--ere measured opt,icall~-t p means of ii polwizing photometer. From there d8t.a ideal exposure conditions BIP quickly defined lor various emulsions and thus the amount of light reaching the photoeleet.ric cell hecomes a direct mcasuro of the exposure time. I n thc application of this met,hod ~everaitests have been made to determine its limitations. From the series of data. collected, an average mean deviation of 5 ~ 6 . 0 %W-RS ohtained under the oonditions of ordinary use. Beenure of the deterioration of the fluorescent material on the screen the unit should be calibrated periodically in order to a8SU1-e sat,isfact.oryperformance. The following advantages may he claimed for the device: consist,ent uniformity of negatives, rmulting in the elimination of repeat exposures; and flesihility, in that the unit is easily ' mhled from readily availsblc parte and may be quickly det:whed for usr in 0 t . 1 lnhorstary ~ prohlemp. ACKNOWLEnCMENT
Ack~onlodgmentis made to Winifrcd Steinorth for assist ance in accumulsting the data in connection with this work. Figure 1. Photometer Unit in Position on Electron Miemscope
As illustrated in Figure 1, the photometer consists essentially of three comoonent Darts: the lens system C. the "hotaclectric probe, P , and the amplifier unit, A .
LmERATURE CITED (1) Bishop, F. IT., Electronics. 23, 11W11 (September 1950). (2) Cuokow, F. W., J . Applied Phys.. 17, GO (1946) (abstract). (3) Hamm. F..4.. I M . . 22,111 (1951)(nhstract). R e c n r ~Jone . ~ 7,1951
