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Isotope fractionation as a source of error in the determination of aldosterone by isotope dilution
787
SHORT COMMUNICATIONS
Isotope fractionation dosterone
as a source of error
in the determination
of al-
by isotope dilution
In an earlier publication’ we described the occurrence of isotope fractionation duringpaperchromato~aphyof [3Hjaldosterone-[W]diacetate*. Theradioactive spot corresponding to this compound was obviously inhomogeneous, as a systematic decrease of the 3H :X ratio was found. The phenomenon of isotope fractionation in adsorption chromatography of doubly-labeled steroids has been describedz-4. Cejka and Vennemanh showed that in partition column chromatography tritium-labeled aldosterone and some of its derivatives partially segregate from the non-tritiated compounds. These authors mention a paper read by Laragh and ~011.8reporting on isotope fractionation when [3H]aldosterone-[14C]diacetate is chromatographed on paper. Recently Kirschner and Lipsett ? described isotope fractionation effects in gasliquid chromatography of steroids containing 3H and r*C. We have extended our observations on this effect to paper chromatography in order to obtain more detailed information on the extent of the isotope fractionation phenomenon and the role it plays, if any, in double-isotope measurements of aldosterone. MATERIALS
AND
METHODS
r,2-[W-d-Aldosterone, 44 C/mmole, acetic r-[Xlanhydride, 10.0 mC/mmole (diluted to 2.0 mC/mmole) and acetic r-[sH]anhydride, 100.0 mC/mmole (diluted to 20.0 mC/mmole) were obtained from the New England Nuclear Corporation (NENC). 4-[%]Aldosterone, 46 mC/mmole, a NENC preparation, was kindly supplied to us by the U.S. National Institutes of Health. After acetylation, the labeled aldosterone diacetate was purified by thin-layer chromatography as previously described I. The purified aldosterone diacetate was chromatographed on paper using the cyclohexane-benzene-methanol-water (6 : 2 : 5 : I) system (IiF 0.65). Free aldosterone was chromatographed on paper using the cyclohexane-benzene-methanol-~vater (5: IO: IO:~) system (& 0.25). Chromatograms were scanned with a Vanguard radioscanner. The scans showed symmetrical peaks of radioactivity without any background on either side. Each peak with its immediately adjacent area was cut into 6 to 8 segments. The segments were eluted and the 3H and 14C radioactivity of the eluates were measured. RESULTS
AND
DISCUSSION
Fig. I shows the distribution of 3H and l*C radioactivity across the peak obtained after chromatography of [3H]aldosterone-[14C]diacetate. The decrease of the 9H/1*Cratio from proximal to distal (in other words, in the direction of the development) is obvious. Table I shows the reproducibility of the isotope fractionation phenomenon, also when different quantities of doubly-labeled steroids are chromatographed and when samples of higher and lower ratios are analysed. Furthermore it will be seen that * In fact, a mixture of [X]
and [Xldiacetates
and of [3H] and [lH]aldosterone C&n. Chiwz. Ada,
was present. 13 (1966) 787-790
788
SHORT
% of the radioactivity of the entire peak
-“C ----
I
7
8
3M disfnt./min/% -
%i
I
/
I
TABLE
I
ISOTOPE
FRACTIOXATION
in paper chromatography
IN PAPER
disint./min
/
6 5 4 3 2 segments of the c~romatograph~c
Fig. r. Isotope fractionation
CO~~~~NICATIONS
CHROMATOGRAPHY
1 peak
of [3H]aldosterone-[rPC]diacetate.
OF [~H]A~DOSTERO~~-~~~~]DIACETAT~
6 disint./min disint./min
[W]entire [W]entire
peak peak
aII/‘4C ratio (disint.~min~ disint. !min) of the entire peak of segment I 2 3 4 2 ;
26418 16 786
42 668 27 803
223630
‘38764
28 302 4032
1.574
I.535
I.612
7.0=9
7.348
2.270
2,424
I.983
2.225 r.75r 1.483 r.rgz r.327
2.276 I.929 I.684 1.381 I.234 I.077 0.987
9.507 8.734 7.485 6.374 4.415 5.023
11.392 9.457 7.727 6.538 5.424 4495 4.022 2.558 7.850 7.142 6.655
1.620 1.33’ I.018 0.981 0.790
Proximal ratio* Central ratio** Distal ratio***
1.672 I.484 I.388
1.680 I.571 I.457
I.706
I.550 1.46~
7.738 7-033 6.802
A Proximal ratio+
i- 6.1% 5.6% - 11.6%
-+ 9.6% + 2.2% - 5.2%
+ 5.9% - 3.9% - 9.4%
+ 10.0% + 1.9% 3.0%
d Central ratio-++ n Distal ratio++
* 3H/14C ratio across the segments
329391 44 829
-i_ 6.9% -- 2.8% - 9.5? 0
523942
69885
7.500 10.271
9.603 8.272 7.287 6.566 5.861 4.997
8.268 7.745 7.Obr i_ 10.2% i- 3.2% 5.8%
r-4.
** 3H/i*C ratio across the segments 3 and 4 (symmetrical
truncation).
*** sH/iaC ratio across the sements 3-6 (ref. 8). + Difference between the ratio across the entire peak and the ratio across the proximal pait of the peak expressed as percentage of the ratio across the entire peak. ++ Analogous Clin. Chim.
with that of d proximal
Acta, 13 (1966) 787-790
ratio.
SHORT COMMUNICATIONS
789
asymmetrical truncation of the chromatographic peak may result in finding a 3H/14C ratio differing from that of the entire peak (0 proximal and d distal ratios varying from + 10.2 to - 11.6%). Symmetrical truncation causes the deviations to be smallest (0 central ratio varying from + 3.2 to - 5.6%). These observations imply that, should asymmetrical truncation be performed with the purpose of eliminating radioactive impurities, such truncation may give rise to considerable errors. To determine whether the position of the tritium label in the molecule is critical, experiments were performed in which the tritium label was in the acetate group (Table II). In this case no significant decrease in the 3H/14C ratio throughout the peak was observed. This implies that, using the chromatographic system under discussion, in double-label studies chromatographing mixtures of aldosterone+H]diacetate and aldosterone-[Xldiacetate-as was done in the original method of Kliman and Petersons-no error is introduced by truncation. TABLE
II
CHROMATOGRAPHY OF MIXTURES OF [sH]- AND [~~C]DIACETATE OF
AND OF [3H]-
AND
UNLABELED
ALDOSTERONE
[14C]~~~~~~~~~~~
[3H]aldosterone [‘PC]aldosterone
Aldosterone-[3H]diacetate Aldosterone-[‘V]diacetate Exfieriment No.
7
8
9
IO
II
disint. /min [3H]entire peak disint. /min [Xlentire peak
I7831 16660
20 108 18022
19131
137 120
‘7 494
38 704
73531 21604
I.070
1.116
I.094
3.543
3.404
I.190 I.153 I.093 1.032 1.052 I.009 0.977
1.256 1.146 I.098 1.106 1.080 1.023 1.167
1.328 1.148 I.147 1.072 1.018 0.978 1.111
5.054 4.673 4.640 4.074 3.339 2.727 I.535 0.554
5.124 4.800 3.869 3.111 1.646 I.327
Proximal ratio* Central ratio Distal ratio
1.060 I.091 I.045
1.102 1.130 1.ogr
I.147 1.176 I.079
4.777 3.567 2.876
4.252 3.493 2.613
d Proximal ratio d Central ratio d Distal ratio
r -
- I. 3 % + 1.3% - 2.2%
+ 4.8% + 7.5% - 1.5%
+ 34.8% + 0.7% _ 19.0%
+ 24.9% 2.6% - 23.2%
$H/14C ratio (disint./min/ disint. /min) of the entire peak of segment I 2 3 4 2 ;
* For definitions
0.9% 1.9% 2.3%
see Table I.
Finally, Table II presents the results obtained after chromatography tures of [3H]- and [14C]aldosterone. It appears that the isotope fractionation
of mixeffect is
even more pronounced than in the case of chromatography of [SH]aldosterone-[‘4C]diacetate (Table I). In the chromatography of the free steroid asymmetrical truncation gives rise to d ratios varying from + 34.8 to - 23.2%. From the studies of Cejka and Venneman s and from our own results it may be expected that tritiated aldosterone segregates from unlabeled aldosterone during paper chromatography. In various double isotope determinations of aldosterone the hormone is purified by paper chroClin. Chim. Acta, 13 (1966) 787-790
SHORT COMMUSICATIONS
790 matography before acetylation Q- 18. In these procedures peak of the tritium-labeled aldosterone may have the specific activity and may give rise to serious errors in cases checking of the extent of the isotope fractionation chromatographic systems is an essential requirement.
asymmetrical cutting of the consequence of altering the the determixlation. In these phenomenon in the various
CONCLUSION
In paper chromatography of [QH]aldosterone-[14C]diacetate, isotope fractionation can give rise to a considerable error in the QH/W ratio, when the chromatographic peak is truncated asymmetrically. In paper chromatography of mixtures of QH- and 1%~labeled aldosterone, the isotope fractionation is even more pronounced. Ihis indicates that, when free aldosterone is chromato~aphed in isotope dilution studies, correct cutting of the spot is even more imperative. In paper chromato~aphy of mixtures of aldosterone-~H]diacetate and aldosterone-[Kjdiacetate isotope fractionation was not observed (in this case, a constant 3H/14Cratio across the peak can function as an index of radiochemical purity). Departme& of Medical and Internal Medicine, University of Nijmegen,
TH.
Biology (The
Netherlands)
J, BENRAAD
M. L. 4. VERWILGHEN P. W. C. KLOPPEKBORG
I TH. J. BENRAAD AND P. W. C. KLOPPENBORG, C&z. Chim. Acta, IZ (1~65) 565. 2 S. ULICK, G. L.NICOLIS AND K.VETTER, in E. E. BAULIEU AIW I?.R~~~~(Eds.),Aldostevone, Blackwell, Oxford, 1964, p. 3. 3 P. I?.KLEIN, Eighth S_~mpos~~~~ on Advawes s’?zTracer ~~~eth~dQlogy,Chicago, 1963. 4 P. D. KLEIN, D. W. SIMBORG AND P. A. SZCZEPANIK, PadreA&S. Chem., 8 (~~64) 357. 5 V. CEJKA AND E.M.VENNEMAN,C&. Chim. Acta, I I (1965) 188. 6 J. H. LARAGH, J, E. SEALEY AND P. I?.KLEIN, Paper read at International ,4tomic Energy Authority Symposion, Salzburg, I 9 6 4. 7 M. ii. KIRSCHNER AND M. R. LIPSETT, J.Lipid. Res., 6(1965) 7. 8 B. KLIMAN AND R. E. PETERSON, J.Biol.Chem., 235 (1960) 1639. 9 H. P. WOLFF AND M. TORBICA, Lancet,i(x~63) 1346. IO K. NEHER, in H. NOWAKOWSIU (Ed.), Aldosteron, Neuntes Symposion der Deutschen Gesellschaft fiir Endokrinologie, Springer-Verlag, Berlin-Gettingen-Heidelberg, 1963, p. 2 I. II B. KLIMAN, Atomlight {New England Nuclear Corp.), 32 (1963). 12 J. S~HWARZ AND R. BLOCH, An%. Eladocrinol. (Paris), 25 (1964) 113. 13 R. E. PETERSON, in E. E. BAULIEU AND P. ROBBL [Eds.), AZdosfevow Blackwell, Oxford, 1964, p. 145.
Received December soth, 1965 Clin. Chim. Acta, 13 (1~66) 787-790
SHORT COMMUNICATIONS
Isotope fractionation dosterone
as a source of error
in the determination
of al-
by isotope dilution
In an earlier publication’ we described the occurrence of isotope fractionation duringpaperchromato~aphyof [3Hjaldosterone-[W]diacetate*. Theradioactive spot corresponding to this compound was obviously inhomogeneous, as a systematic decrease of the 3H :X ratio was found. The phenomenon of isotope fractionation in adsorption chromatography of doubly-labeled steroids has been describedz-4. Cejka and Vennemanh showed that in partition column chromatography tritium-labeled aldosterone and some of its derivatives partially segregate from the non-tritiated compounds. These authors mention a paper read by Laragh and ~011.8reporting on isotope fractionation when [3H]aldosterone-[14C]diacetate is chromatographed on paper. Recently Kirschner and Lipsett ? described isotope fractionation effects in gasliquid chromatography of steroids containing 3H and r*C. We have extended our observations on this effect to paper chromatography in order to obtain more detailed information on the extent of the isotope fractionation phenomenon and the role it plays, if any, in double-isotope measurements of aldosterone. MATERIALS
AND
METHODS
r,2-[W-d-Aldosterone, 44 C/mmole, acetic r-[Xlanhydride, 10.0 mC/mmole (diluted to 2.0 mC/mmole) and acetic r-[sH]anhydride, 100.0 mC/mmole (diluted to 20.0 mC/mmole) were obtained from the New England Nuclear Corporation (NENC). 4-[%]Aldosterone, 46 mC/mmole, a NENC preparation, was kindly supplied to us by the U.S. National Institutes of Health. After acetylation, the labeled aldosterone diacetate was purified by thin-layer chromatography as previously described I. The purified aldosterone diacetate was chromatographed on paper using the cyclohexane-benzene-methanol-water (6 : 2 : 5 : I) system (IiF 0.65). Free aldosterone was chromatographed on paper using the cyclohexane-benzene-methanol-~vater (5: IO: IO:~) system (& 0.25). Chromatograms were scanned with a Vanguard radioscanner. The scans showed symmetrical peaks of radioactivity without any background on either side. Each peak with its immediately adjacent area was cut into 6 to 8 segments. The segments were eluted and the 3H and 14C radioactivity of the eluates were measured. RESULTS
AND
DISCUSSION
Fig. I shows the distribution of 3H and l*C radioactivity across the peak obtained after chromatography of [3H]aldosterone-[14C]diacetate. The decrease of the 9H/1*Cratio from proximal to distal (in other words, in the direction of the development) is obvious. Table I shows the reproducibility of the isotope fractionation phenomenon, also when different quantities of doubly-labeled steroids are chromatographed and when samples of higher and lower ratios are analysed. Furthermore it will be seen that * In fact, a mixture of [X]
and [Xldiacetates
and of [3H] and [lH]aldosterone C&n. Chiwz. Ada,
was present. 13 (1966) 787-790
788
SHORT
% of the radioactivity of the entire peak
-“C ----
I
7
8
3M disfnt./min/% -
%i
I
/
I
TABLE
I
ISOTOPE
FRACTIOXATION
in paper chromatography
IN PAPER
disint./min
/
6 5 4 3 2 segments of the c~romatograph~c
Fig. r. Isotope fractionation
CO~~~~NICATIONS
CHROMATOGRAPHY
1 peak
of [3H]aldosterone-[rPC]diacetate.
OF [~H]A~DOSTERO~~-~~~~]DIACETAT~
6 disint./min disint./min
[W]entire [W]entire
peak peak
aII/‘4C ratio (disint.~min~ disint. !min) of the entire peak of segment I 2 3 4 2 ;
26418 16 786
42 668 27 803
223630
‘38764
28 302 4032
1.574
I.535
I.612
7.0=9
7.348
2.270
2,424
I.983
2.225 r.75r 1.483 r.rgz r.327
2.276 I.929 I.684 1.381 I.234 I.077 0.987
9.507 8.734 7.485 6.374 4.415 5.023
11.392 9.457 7.727 6.538 5.424 4495 4.022 2.558 7.850 7.142 6.655
1.620 1.33’ I.018 0.981 0.790
Proximal ratio* Central ratio** Distal ratio***
1.672 I.484 I.388
1.680 I.571 I.457
I.706
I.550 1.46~
7.738 7-033 6.802
A Proximal ratio+
i- 6.1% 5.6% - 11.6%
-+ 9.6% + 2.2% - 5.2%
+ 5.9% - 3.9% - 9.4%
+ 10.0% + 1.9% 3.0%
d Central ratio-++ n Distal ratio++
* 3H/14C ratio across the segments
329391 44 829
-i_ 6.9% -- 2.8% - 9.5? 0
523942
69885
7.500 10.271
9.603 8.272 7.287 6.566 5.861 4.997
8.268 7.745 7.Obr i_ 10.2% i- 3.2% 5.8%
r-4.
** 3H/i*C ratio across the segments 3 and 4 (symmetrical
truncation).
*** sH/iaC ratio across the sements 3-6 (ref. 8). + Difference between the ratio across the entire peak and the ratio across the proximal pait of the peak expressed as percentage of the ratio across the entire peak. ++ Analogous Clin. Chim.
with that of d proximal
Acta, 13 (1966) 787-790
ratio.
SHORT COMMUNICATIONS
789
asymmetrical truncation of the chromatographic peak may result in finding a 3H/14C ratio differing from that of the entire peak (0 proximal and d distal ratios varying from + 10.2 to - 11.6%). Symmetrical truncation causes the deviations to be smallest (0 central ratio varying from + 3.2 to - 5.6%). These observations imply that, should asymmetrical truncation be performed with the purpose of eliminating radioactive impurities, such truncation may give rise to considerable errors. To determine whether the position of the tritium label in the molecule is critical, experiments were performed in which the tritium label was in the acetate group (Table II). In this case no significant decrease in the 3H/14C ratio throughout the peak was observed. This implies that, using the chromatographic system under discussion, in double-label studies chromatographing mixtures of aldosterone+H]diacetate and aldosterone-[Xldiacetate-as was done in the original method of Kliman and Petersons-no error is introduced by truncation. TABLE
II
CHROMATOGRAPHY OF MIXTURES OF [sH]- AND [~~C]DIACETATE OF
AND OF [3H]-
AND
UNLABELED
ALDOSTERONE
[14C]~~~~~~~~~~~
[3H]aldosterone [‘PC]aldosterone
Aldosterone-[3H]diacetate Aldosterone-[‘V]diacetate Exfieriment No.
7
8
9
IO
II
disint. /min [3H]entire peak disint. /min [Xlentire peak
I7831 16660
20 108 18022
19131
137 120
‘7 494
38 704
73531 21604
I.070
1.116
I.094
3.543
3.404
I.190 I.153 I.093 1.032 1.052 I.009 0.977
1.256 1.146 I.098 1.106 1.080 1.023 1.167
1.328 1.148 I.147 1.072 1.018 0.978 1.111
5.054 4.673 4.640 4.074 3.339 2.727 I.535 0.554
5.124 4.800 3.869 3.111 1.646 I.327
Proximal ratio* Central ratio Distal ratio
1.060 I.091 I.045
1.102 1.130 1.ogr
I.147 1.176 I.079
4.777 3.567 2.876
4.252 3.493 2.613
d Proximal ratio d Central ratio d Distal ratio
r -
- I. 3 % + 1.3% - 2.2%
+ 4.8% + 7.5% - 1.5%
+ 34.8% + 0.7% _ 19.0%
+ 24.9% 2.6% - 23.2%
$H/14C ratio (disint./min/ disint. /min) of the entire peak of segment I 2 3 4 2 ;
* For definitions
0.9% 1.9% 2.3%
see Table I.
Finally, Table II presents the results obtained after chromatography tures of [3H]- and [14C]aldosterone. It appears that the isotope fractionation
of mixeffect is
even more pronounced than in the case of chromatography of [SH]aldosterone-[‘4C]diacetate (Table I). In the chromatography of the free steroid asymmetrical truncation gives rise to d ratios varying from + 34.8 to - 23.2%. From the studies of Cejka and Venneman s and from our own results it may be expected that tritiated aldosterone segregates from unlabeled aldosterone during paper chromatography. In various double isotope determinations of aldosterone the hormone is purified by paper chroClin. Chim. Acta, 13 (1966) 787-790
SHORT COMMUSICATIONS
790 matography before acetylation Q- 18. In these procedures peak of the tritium-labeled aldosterone may have the specific activity and may give rise to serious errors in cases checking of the extent of the isotope fractionation chromatographic systems is an essential requirement.
asymmetrical cutting of the consequence of altering the the determixlation. In these phenomenon in the various
CONCLUSION
In paper chromatography of [QH]aldosterone-[14C]diacetate, isotope fractionation can give rise to a considerable error in the QH/W ratio, when the chromatographic peak is truncated asymmetrically. In paper chromatography of mixtures of QH- and 1%~labeled aldosterone, the isotope fractionation is even more pronounced. Ihis indicates that, when free aldosterone is chromato~aphed in isotope dilution studies, correct cutting of the spot is even more imperative. In paper chromato~aphy of mixtures of aldosterone-~H]diacetate and aldosterone-[Kjdiacetate isotope fractionation was not observed (in this case, a constant 3H/14Cratio across the peak can function as an index of radiochemical purity). Departme& of Medical and Internal Medicine, University of Nijmegen,
TH.
Biology (The
Netherlands)
J, BENRAAD
M. L. 4. VERWILGHEN P. W. C. KLOPPEKBORG
I TH. J. BENRAAD AND P. W. C. KLOPPENBORG, C&z. Chim. Acta, IZ (1~65) 565. 2 S. ULICK, G. L.NICOLIS AND K.VETTER, in E. E. BAULIEU AIW I?.R~~~~(Eds.),Aldostevone, Blackwell, Oxford, 1964, p. 3. 3 P. I?.KLEIN, Eighth S_~mpos~~~~ on Advawes s’?zTracer ~~~eth~dQlogy,Chicago, 1963. 4 P. D. KLEIN, D. W. SIMBORG AND P. A. SZCZEPANIK, PadreA&S. Chem., 8 (~~64) 357. 5 V. CEJKA AND E.M.VENNEMAN,C&. Chim. Acta, I I (1965) 188. 6 J. H. LARAGH, J, E. SEALEY AND P. I?.KLEIN, Paper read at International ,4tomic Energy Authority Symposion, Salzburg, I 9 6 4. 7 M. ii. KIRSCHNER AND M. R. LIPSETT, J.Lipid. Res., 6(1965) 7. 8 B. KLIMAN AND R. E. PETERSON, J.Biol.Chem., 235 (1960) 1639. 9 H. P. WOLFF AND M. TORBICA, Lancet,i(x~63) 1346. IO K. NEHER, in H. NOWAKOWSIU (Ed.), Aldosteron, Neuntes Symposion der Deutschen Gesellschaft fiir Endokrinologie, Springer-Verlag, Berlin-Gettingen-Heidelberg, 1963, p. 2 I. II B. KLIMAN, Atomlight {New England Nuclear Corp.), 32 (1963). 12 J. S~HWARZ AND R. BLOCH, An%. Eladocrinol. (Paris), 25 (1964) 113. 13 R. E. PETERSON, in E. E. BAULIEU AND P. ROBBL [Eds.), AZdosfevow Blackwell, Oxford, 1964, p. 145.
Received December soth, 1965 Clin. Chim. Acta, 13 (1~66) 787-790