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A source of error in the use of radioactive substrates for metabolic studies
LETTERS
TO
2. FRAENKEL-CONRAT,H.,BEAN,R.!%,.4m
H.,
WEAVER,
3. TISRMINIELLO, NORD,
J. Viol.
THE
JIINE-
Chenl.
177, 385 (1949).
L., SRI RAM, J., BIER, F. F., Arch. B&hem. Biophys.
%I.,
AND
67, 252
(1955). 4. CRAKI,
Z.,
TERMINIELLO,
F. F., Arch.
XORD,
Department of Organic Chernixtry and Enaynolog?J3 Fordham versity
6. 1)1x0x, Chem.
WOOD,
H. N.,
H., AND NELJRATH, 226, 1049 (1957).
6.
A Source
of
TERESA
THER.4TTIL-.kNTONY'
hf. BIER F. F. r\;ORD
Un,i-
L., BIER, M., AND Biochem. Bioph,ys. 69, 644
(1957). 5. JJALLS, A. K., AND 219, 245 (1956).
1.51
EDITORS
Error
in the
J. Viol. H.,
Use
Received Januar:J 15, 1960
(‘hem. J.
Biol.
try,
of Radioactive
Recently values have been published (I) for the radiochemical yields of Cl402 from metabolism of glucose-l-Cl4 (G-I-Cll) and G-6-C14 by electric tissue of E’lectrophorus electricas, as well as by a number of other tissues, and for the ratios Cl402 from G6-C14/C140? from G-l-C14. A later paper (2) describes the effect,s of certain compounds on these same values from slices of guinea pig brain cortex. Subsequent experiments indicat)e a source of possible error which was not previously suspected and which, under certain conditions, may require correct,ions in t,he interpretation of data. This is shown in the following experiments, t,ypical of several recently performed. Aliquots of G-l-Cl4 and G-6-C’,*, obtained commercially and used wit,hout further purification, were incubated with t.hin strips of Sachs organ or slices of main organ from the elect,ric fish. Simultaneously identical samples of t,he radioactive glucose wrre incubated with buffer only. The condt,ions are given in Table I. In summary, the mdioactive glucose in all experiments RTas in contact with aqueous buffer at 30°C. for 3 hr., pH 7.2-7.3, for an additional 30 min.-l hr. in approximatelyO.2 .V H$Or Finally, the radioactivity, trapped in 0.125 ml. 20% KOH, was assayed by the method of Van Slykr: and co-workers (3)2. Table I, whichgivcs i he results corrcctetl to absolute disintrgrat,ions per minute after subtraction of background, shows a relatively large spontaneous production of rudioactivit!. from G-1-C” and a smaller but appreciable production of radioactivity from G-6-C11. The data in the right-hand column of Table I were * This work was supported by the Division of Itesearch Grants and Fellowships, National Institutes of Health, Grant No. 11-740, U. S. Public IIenlth Service. 2 Itefcrc~nces (I, 6, 7) of this reference together with this reference give all information for i hc assav of C!“.
2 Permanent address : Department Delhi University, Xew Delhi, B Communication No. 360. Substrates
for
Metabolic
of ChemisIndia.
Studies’
then treated in two different ways, namely, as total apparent yields from tissue, and as yields from tissue minus yields due to spontaneous release of radioactivity, i.e., minus controls. Bfter correct,ions for the different, amounts of tissue and the different specific activities, the results are given in Table II. These values indicate that, although the ratio CIWy from G-6-C14/C*40~ from G-l-Cl4 st,ill remains small for electric tissue, the radiochemical yields at the low level encountered in t,his tissue may be much lower than previous1.v reported (1). TABI,E
I
Tissue
dzsinlcgraiionsjn~in.
Sachs Sachs Main Main None (control) KOIlC
(control)
1 t
I c:-6-C'1
/ 1,H'I6,OO(I~
123 I
I I u Vessel contained either 200-300 mg. tissue and 2.5 ml. buffer or no tissue and 2.75 ml. buffer of the following millimolar composition: NaCl, 160; KCl, 5; NasHI’ , I; NaHzPO4.He0, 0.3; CnCl?.2HzO, 2; fifgCl~.GHaO, 2; equilibration with O:, ; pH adjusted to 7.2-7.3 with sodium phosphate. Substrate, 25 rmoles glucose per vessel (radioactive plus nonradioactive). Incubation, 3 hr., 30°C.; atmosphere, 02 . After 3 hr., 0.125 ml. 5 X H,SO4 added from side-arm.
152
LETTERS
TABLE METABOLISM
II
OF GL1XOSE-C'4
BY Source of radiochemical yield
ELECTRIC
TISSUW
Radiochemical
yield
from
-__ [
G-1-C’”
Sachs Sachs minus cont,rol Main ?vI:tin minus control
TO
Gm6-c,”
1 G-h-C’4 , (;-‘-c’4
%
0;
6.46 X lOP* 1.89 x lo-*<
0.67 X 10-Z 0.22 x 10-z
0. 104 0.116
6.70 1.20
0.71 x 0.15 x
0.106 0.125
x x
lo-* IO-2
10-z IO-2
a Data from Table I treated as described in text. Radiochemical yields calculated for 500 mg. fresh tissue. * 1940 x 500 x 100 = 6.46 x 10-z “/b 5,372,OOO X 279 c (1940 - 1373) x 500 x 100 = , 8. x I(l~e 7 . 2 0 5,372,OOO X 279 For those tissues having considerably higher yields of C1% (brain and diaphragm), the error due t,o spontaneous release of radioactivit,y becomes smaller. For example, high radiochemical yields of 2% from G-l-U4 and <;-6-C” would become 1.75% and 1.98%, respeci.ively, and the rat,io would change only slightly, from 1.0 to 1.1. For potassium-stimulated metabolism (4) of guinea pig brain cortex with :t radiochemical yield (per 500 mg. fresh t,issue) of about 10% and for the much higher yields of C’*OY from G-l-U4 under certain chemical stimulation (2), the error due to spontaneous production of radioactivit,v becomes negligible. However, it is not possible t,o correct UCCIP rately results obtained previously at the low levels of radiochemical yield (0.270 and below) because, in retrospect, it. appears that not all batjches of commercial glucose-c” gave the same spontaneous production of C I4 in some volatile form. This may, in part, account for the rather wide variations sometimes found in investigations of this kind. Methylation
of
2-Hydroxyestradiol-17/z?
In 1957, 2.methoxycstronc was identified as a normal metabolite of cst,radiol-17p in the human (l-3). To explain t.his finding, a two-step mechanism involving 2-hydroxyestradiol and its subse1 This work was support,cd in part by Grants A-1078 and A-767 from the National Inst,itutc of Arthritis and Metabolic l)iseases, Natjional Instit,utes of Hcalt,h, Public Health Services.
THE
KDITORS
As to the nature of the Cl4 which has been spontaneously released from G-l-Cl4 and, to a smaller extent, from G-6-U4, it is either a volatile impurity or a decomposition of about O.Ol’% extent for G-l-CP4 and much less (0.001-0.0005~0) for G-6-P. If an impurity, it has been quite undet&able by the usual test.s (chromatography, synt)hesis of derivatives, etc.) which this investigator and others have employed. Furthermore, to t)e volatile from acid medium and capable of passing a dry-ice I r‘ap (3) would almost limit the impurity t,o some form of carbon dioxide. Chemical decomposition in the aqueous medium of the present cxpcriments stems unlikely due to inconsistencies from b:ttjch to batch of glucose-C14. There is also the possibility of radiation decomposition (5, 6) before use, or even before the solution and freezing of each 50~~. sample in 2.0 ml. water. Although t,he samples were not accumulated in this laboratory, they may have had a prior storage history. E~FERENCES
1. WOSKIX, F. C. G., Arch. Riochem. Nioph!ys. 86, 141 (1959). 2. HOSKIN, F. C. G., Riochinr. et &oph[js. A&L, in press. 3. SINEX, F. ;\I., I'LAZIN, J., CLAREIJS, I>., BERNSTEIS, W., VAN SLYKE, 1). I)., AND CHASE, R., .I. Hiol. C’hem. 213, 673 (1955). 4. GHOSH, J. J., AND QIJASTEL, J. H., Nature 174, 28 (1954). 5. TOLRERT, 13. bl., ADAMS, P. T., BENNETT, R. I,., It.
HI-GHES, A. RI., KIRK, Al. R., RI., SOLLER, R. &I., OSTWALD,
CALVIN,
Xl.,
J.
Am.
Chem.
TAXWON, It., .4NII
Sot.
76,
1867
(1953). 6. B.~KER, N., GIBBONS, ii. A., Hiochim. ct Riophys.
I'.,
Deparhmt oj ~Veurolog~y College of Physicians and Surgeons Colwnbia Vniversity New Fork, New York Receioed Janrraq 5, 1960 To 2-Methoxyestrone
in
AXI)
Acta 1'.
the
?MPI,EY,
Et.
28, 579 (1958). C.
G.
HOSKIN
Human’
quent methylat,ion was proposed (3). However, on the basis of the evidence available at the time, a methoxylation reaction could not be excluded, since no 0-hydroxy phenolic steroids wcrc isolated. The obvious importance of 2-methoxyest,rone led us to investigate t,he mechanism whereby cstradiol was mctnbolized to this compound. In a recent communication (4) we reported the synthesis of 2-hydrns~estradiol-17~. This compound has
TO
2. FRAENKEL-CONRAT,H.,BEAN,R.!%,.4m
H.,
WEAVER,
3. TISRMINIELLO, NORD,
J. Viol.
THE
JIINE-
Chenl.
177, 385 (1949).
L., SRI RAM, J., BIER, F. F., Arch. B&hem. Biophys.
%I.,
AND
67, 252
(1955). 4. CRAKI,
Z.,
TERMINIELLO,
F. F., Arch.
XORD,
Department of Organic Chernixtry and Enaynolog?J3 Fordham versity
6. 1)1x0x, Chem.
WOOD,
H. N.,
H., AND NELJRATH, 226, 1049 (1957).
6.
A Source
of
TERESA
THER.4TTIL-.kNTONY'
hf. BIER F. F. r\;ORD
Un,i-
L., BIER, M., AND Biochem. Bioph,ys. 69, 644
(1957). 5. JJALLS, A. K., AND 219, 245 (1956).
1.51
EDITORS
Error
in the
J. Viol. H.,
Use
Received Januar:J 15, 1960
(‘hem. J.
Biol.
try,
of Radioactive
Recently values have been published (I) for the radiochemical yields of Cl402 from metabolism of glucose-l-Cl4 (G-I-Cll) and G-6-C14 by electric tissue of E’lectrophorus electricas, as well as by a number of other tissues, and for the ratios Cl402 from G6-C14/C140? from G-l-C14. A later paper (2) describes the effect,s of certain compounds on these same values from slices of guinea pig brain cortex. Subsequent experiments indicat)e a source of possible error which was not previously suspected and which, under certain conditions, may require correct,ions in t,he interpretation of data. This is shown in the following experiments, t,ypical of several recently performed. Aliquots of G-l-Cl4 and G-6-C’,*, obtained commercially and used wit,hout further purification, were incubated with t.hin strips of Sachs organ or slices of main organ from the elect,ric fish. Simultaneously identical samples of t,he radioactive glucose wrre incubated with buffer only. The condt,ions are given in Table I. In summary, the mdioactive glucose in all experiments RTas in contact with aqueous buffer at 30°C. for 3 hr., pH 7.2-7.3, for an additional 30 min.-l hr. in approximatelyO.2 .V H$Or Finally, the radioactivity, trapped in 0.125 ml. 20% KOH, was assayed by the method of Van Slykr: and co-workers (3)2. Table I, whichgivcs i he results corrcctetl to absolute disintrgrat,ions per minute after subtraction of background, shows a relatively large spontaneous production of rudioactivit!. from G-1-C” and a smaller but appreciable production of radioactivity from G-6-C11. The data in the right-hand column of Table I were * This work was supported by the Division of Itesearch Grants and Fellowships, National Institutes of Health, Grant No. 11-740, U. S. Public IIenlth Service. 2 Itefcrc~nces (I, 6, 7) of this reference together with this reference give all information for i hc assav of C!“.
2 Permanent address : Department Delhi University, Xew Delhi, B Communication No. 360. Substrates
for
Metabolic
of ChemisIndia.
Studies’
then treated in two different ways, namely, as total apparent yields from tissue, and as yields from tissue minus yields due to spontaneous release of radioactivity, i.e., minus controls. Bfter correct,ions for the different, amounts of tissue and the different specific activities, the results are given in Table II. These values indicate that, although the ratio CIWy from G-6-C14/C*40~ from G-l-Cl4 st,ill remains small for electric tissue, the radiochemical yields at the low level encountered in t,his tissue may be much lower than previous1.v reported (1). TABI,E
I
Tissue
dzsinlcgraiionsjn~in.
Sachs Sachs Main Main None (control) KOIlC
(control)
1 t
I c:-6-C'1
/ 1,H'I6,OO(I~
123 I
I I u Vessel contained either 200-300 mg. tissue and 2.5 ml. buffer or no tissue and 2.75 ml. buffer of the following millimolar composition: NaCl, 160; KCl, 5; NasHI’ , I; NaHzPO4.He0, 0.3; CnCl?.2HzO, 2; fifgCl~.GHaO, 2; equilibration with O:, ; pH adjusted to 7.2-7.3 with sodium phosphate. Substrate, 25 rmoles glucose per vessel (radioactive plus nonradioactive). Incubation, 3 hr., 30°C.; atmosphere, 02 . After 3 hr., 0.125 ml. 5 X H,SO4 added from side-arm.
152
LETTERS
TABLE METABOLISM
II
OF GL1XOSE-C'4
BY Source of radiochemical yield
ELECTRIC
TISSUW
Radiochemical
yield
from
-__ [
G-1-C’”
Sachs Sachs minus cont,rol Main ?vI:tin minus control
TO
Gm6-c,”
1 G-h-C’4 , (;-‘-c’4
%
0;
6.46 X lOP* 1.89 x lo-*<
0.67 X 10-Z 0.22 x 10-z
0. 104 0.116
6.70 1.20
0.71 x 0.15 x
0.106 0.125
x x
lo-* IO-2
10-z IO-2
a Data from Table I treated as described in text. Radiochemical yields calculated for 500 mg. fresh tissue. * 1940 x 500 x 100 = 6.46 x 10-z “/b 5,372,OOO X 279 c (1940 - 1373) x 500 x 100 = , 8. x I(l~e 7 . 2 0 5,372,OOO X 279 For those tissues having considerably higher yields of C1% (brain and diaphragm), the error due t,o spontaneous release of radioactivit,y becomes smaller. For example, high radiochemical yields of 2% from G-l-U4 and <;-6-C” would become 1.75% and 1.98%, respeci.ively, and the rat,io would change only slightly, from 1.0 to 1.1. For potassium-stimulated metabolism (4) of guinea pig brain cortex with :t radiochemical yield (per 500 mg. fresh t,issue) of about 10% and for the much higher yields of C’*OY from G-l-U4 under certain chemical stimulation (2), the error due to spontaneous production of radioactivit,v becomes negligible. However, it is not possible t,o correct UCCIP rately results obtained previously at the low levels of radiochemical yield (0.270 and below) because, in retrospect, it. appears that not all batjches of commercial glucose-c” gave the same spontaneous production of C I4 in some volatile form. This may, in part, account for the rather wide variations sometimes found in investigations of this kind. Methylation
of
2-Hydroxyestradiol-17/z?
In 1957, 2.methoxycstronc was identified as a normal metabolite of cst,radiol-17p in the human (l-3). To explain t.his finding, a two-step mechanism involving 2-hydroxyestradiol and its subse1 This work was support,cd in part by Grants A-1078 and A-767 from the National Inst,itutc of Arthritis and Metabolic l)iseases, Natjional Instit,utes of Hcalt,h, Public Health Services.
THE
KDITORS
As to the nature of the Cl4 which has been spontaneously released from G-l-Cl4 and, to a smaller extent, from G-6-U4, it is either a volatile impurity or a decomposition of about O.Ol’% extent for G-l-CP4 and much less (0.001-0.0005~0) for G-6-P. If an impurity, it has been quite undet&able by the usual test.s (chromatography, synt)hesis of derivatives, etc.) which this investigator and others have employed. Furthermore, to t)e volatile from acid medium and capable of passing a dry-ice I r‘ap (3) would almost limit the impurity t,o some form of carbon dioxide. Chemical decomposition in the aqueous medium of the present cxpcriments stems unlikely due to inconsistencies from b:ttjch to batch of glucose-C14. There is also the possibility of radiation decomposition (5, 6) before use, or even before the solution and freezing of each 50~~. sample in 2.0 ml. water. Although t,he samples were not accumulated in this laboratory, they may have had a prior storage history. E~FERENCES
1. WOSKIX, F. C. G., Arch. Riochem. Nioph!ys. 86, 141 (1959). 2. HOSKIN, F. C. G., Riochinr. et &oph[js. A&L, in press. 3. SINEX, F. ;\I., I'LAZIN, J., CLAREIJS, I>., BERNSTEIS, W., VAN SLYKE, 1). I)., AND CHASE, R., .I. Hiol. C’hem. 213, 673 (1955). 4. GHOSH, J. J., AND QIJASTEL, J. H., Nature 174, 28 (1954). 5. TOLRERT, 13. bl., ADAMS, P. T., BENNETT, R. I,., It.
HI-GHES, A. RI., KIRK, Al. R., RI., SOLLER, R. &I., OSTWALD,
CALVIN,
Xl.,
J.
Am.
Chem.
TAXWON, It., .4NII
Sot.
76,
1867
(1953). 6. B.~KER, N., GIBBONS, ii. A., Hiochim. ct Riophys.
I'.,
Deparhmt oj ~Veurolog~y College of Physicians and Surgeons Colwnbia Vniversity New Fork, New York Receioed Janrraq 5, 1960 To 2-Methoxyestrone
in
AXI)
Acta 1'.
the
?MPI,EY,
Et.
28, 579 (1958). C.
G.
HOSKIN
Human’
quent methylat,ion was proposed (3). However, on the basis of the evidence available at the time, a methoxylation reaction could not be excluded, since no 0-hydroxy phenolic steroids wcrc isolated. The obvious importance of 2-methoxyest,rone led us to investigate t,he mechanism whereby cstradiol was mctnbolized to this compound. In a recent communication (4) we reported the synthesis of 2-hydrns~estradiol-17~. This compound has