Childrm’s Hospital Oakland Research lnnstitute,Oakland, CA, USA A 1935 issue of the journat S&MY contains the first report of tracer methodofogy using an isotopicalfy fabeled precursor, and it i~cfuded work on steroids. Schoenheimer and Rittenberg showed that administration of partially iabeted coprostanone (4,.5-ZHz) to humans resulted in fecaJ excretion of labeled coprostanoI.* For the hrst time, precursors and products could be unequivocally linked and metabolism could be studied in a dynamic mode.
Coprostanone
Copfostanol
The need for precursor labeling had long been recognized and had been attempted using easily detectable adducts with benzene or halides, so the potential use of heavy isotopes was evident immediately after their discovery. This explains why only 3 years were to elapse between Urey’s discovery of deuterium’ and the first publication on the use of deuterium-IabeIed precursors in metabolic studies. The potential use of isotopes of other eIements present in biotogic systems was also recognized. Sulfur isotopes, i3C, isO, and lSN had all been isotated in the period from 1927 to 1930, and the first use of r3C in metabolism studies was that of Wood et al. ,? who demonstrated carbon dioxide fixation by bacteria grown in NaHi3C03 solutions. The i3C content of propionic, succinic, malic, and fumaric acids was determined using a “mass spectrograph.” it had been shown early that use of stable isotopelabeled compounds in intact organisms was probably safe. In 1933, Taylor et al.” had shown that fish and earthworms could survive in predominantly heavy water.* The early research on stable isotope use was spearheaded by Riftenb~rg and Schoenheimer at Co-
* For a later and more complete discussion of the toxicity of deuterium in mamm~s, the reader should consuit T~~mpson,s Address reprint requests to Dr. Cedric H. L. Shackleton, Children’s Hospital Oakland Research Institute, 747 2nd Street, Oakland, CA 94609. USA.
0 1990 Butterworth
Publishers
lumbia university, fater to be joined by Konrad Bfoch. Bloch and his coffeagues must be credited with leading steroid biosynthesis investigations during the 1940s and 1950s. An early example of steroid metabolic studies was their demonstration in 1943 that labeled cholic acid (a bile acid) was synthesized from labeled cholesterol by dogs.” In the first reported ~~~u~ expe~ment, Bfoch administered labeled cholesterol to a woman in the eighth month of pregnancy and isolated labeled pregnanedio1.7 These experiments showed for the first time that in the mammalian system, hormonal steroids and bile acids had choiesterof as a common precursor. If is remarkable that the first human experiment was on a pregnant woman; one senses that institutional review boards or their predecessors were non-existent in previous times. Of course, we now know that it is precisely in these sensitive situations (pregnancy or the newborn) that stable isotope labeling in metabolic studies should be encouraged for reasons of safety. One of the fnnd~ental early experiments in steroid biosynthesis was carried out by Bfoch and coworkers, who showed that cholesterof could be formed from deuterated or j3C-labeled acetate.g AII of these studies illustrate the fruitfulness of the period from 1935 to 1945 in biosynthetic studies using stable isotopes. Tracer methodology took a big Ieap after the war with the availability of i4C, which then became the focus for production of labeled precursors. For the next two decades, the use of 14C and 3H completely overshadowed the use of stable isotope-labeled materials. The decline of stable isotope work may be attributed to the unavailability of good reliable instrumentation for determination of isotope incorporation. A few mass spectrometers were available, but they were unreIiable and di~~ult to use and maintain. In contrast, by the late t9SOs, liquid scintillation counting had simplified detection of radioisotopes to the extent that novices could be readily trained in their use, In the haste to abandon stable isotope studies, it was frequently forgotten that the use of stable and radiolabeling techniques are complementary and that different information could be obtained. While radioactive measurement would tell you whether the product was Iabeled, it could not readily give info~ation on the position of the label. The CompIementary nature of the two techniques was weli-illustrated in the pioneering work on cholesterol synthesis from acetate via squalene carSteroids,
1990, vol. 55, April
139
I96Os, interest centered on studying biosynthetic pathways, which was most readily carried out with radiolabels. In contrast, studies performed during the 1970s and 1980s have focused to a greater extent on mechanistic aspects of biochemical t~nsfo~~tion~, in which information was required on the origin or f&e of’ specific atoms in a particular molecule. Interest in this field has been driven by pharmaceutical research, in which the conversion and breakdown of drugs must be accurately determined. Finally, one must mention the increased awareness of safety which has limited the widespread use of radiolabels in human metabolism studies. Investigations on the sequence of aromatization carried out by the late Lewis Engel and his group could be seen as introducing a new era of stable isotope studies. In a novel but simple experiment, they isolated several of the intermediates in placental microsomal estrogen formation.” By using as substrates testosterone or androstenedione, which were about 50% monodeuterated, all intermediates and the estrogen products were easily distinguishable by having M+ and [M + 11” of approximateiy equal size in their mass spectra. They termed this methodology the “twinion” technique. Figure i shows the spectra of isolated and reference estrone demonstrating the efficacy of this method. The current utilization of stable isotopes for in vivo steroid metabolism and turnover studies
ried out by Bloch.” By the judicious USC of acetate labeled in the one or two positions with ‘“C, or with a mixture of ‘“C and ‘IC, they were able to report in many cases which carbon atom of acetate gave rise to which carbon atom in choiestcrol. In their later experiment using squalene synthesized from 3-r3C acetoacetate and 4..rRC acetoacetate, they were able to determine exactly the mechanism of cyclization of squalene to lanosterol. The lanosterol had to be converted to ethylene for which the proportions of mono-‘%?, di“C, and unlabeled r3C could be determined.“’ The respective qualities of stable isotopes and radioisotopes in studies of metabolism have been summarized in Table 1. This table emphasizes the different roles that each plays, suggesting that with equal availability of suitable instrumentation for their measurement, both should be used. No single factor was responsible for the resurgence of interest in stable isotopes seen over the last few years. On one side was the increased availability of the isotopes themselves in high degrees of purity and an enlarging literature on how to introduce them into the steroid molecule.‘r and on another was the deveiopment and commercialization of mass spectrometers and nuclear magnetic resonance (NMR) instruments with which to detect them within biomolecules. The need for stable isotope studies was also widely recognized. With a few notable exceptions, in the 1950s and ftm 90.
iv+
73
342
\
BO-
COMPOUND
70-
ISOLATED
SO-
216 !
SOUOJO-
257 \
75 ./
327
10 0 low 90.
M' 342
73
BO-
0
70
uo-
MW
30.
342
244 232
I
M-28
i llll 60
60
100
120
I 1uo
II 160
II 180
II
III 200
I 220
II
‘8:
II 2uo
I 260
4
*St 280
.L 300
“,;:”
3j4
A
320
i
,.1. 31ia
ii.
360
M/E Figure 1 Mass spectra of trimethylsilyl ethers of estrone isolated from incubation of [7@+l]testosterone placental microsomes (top) and of the reference compound (bottom). (Reprinted with permission.lzl
140
Steroids,
1990, vol. 55, April
(42 atom % excess) with
Introduction: Table 1 Comparative features of stable and radioisotope topes in biochemical investigation Radiolabeled Can all common biologic elements be studied? Position of label in product easily discernable? Influence on substrate/product pool size Can total recovery be determined readily? Sensitivity of measurement Ease of measurement Use limited by ethical considerations? Suitability for use in enzyme mechanism studies Lower cost per experiment? Can be used for quantification? Abbreviations: tography-mass
No
iso-
Stable isotope-labeled
Shackleton
munoassays. In spite of greater availability of stable isotope-labeled steroids and better methods for determining their presence in compounds isolated from incubates or body fluids (e.g., NMR, high-pressure liquid chromatography-mass spectrometry, GUMS), their use remains severely limited. While stable isotopes are not appropriate for every investigation, I hope that the experimentation described in these pages will encourage more investigators to consider their use.
No
Yes (N and 0 have only stable isotope forms) Yes
None
Moderate
Yes
No
I.
High
2.
Easy Yes
Low (except for known metabolites) More difficult No
Low
High
4.
Yes
No
5.
Yes (RIA)
Yes (GUMS)
6.
References
RIA, radioimmunoassay; spectrometry.
GUMS,
gas chroma-
3.
I. 8. 9.
can be traced to the bile acid studies of Watkins et aLi and the steroid sulfate metabolism studies by Baillie et aLi Both of these investigations were carried out in situations in which permission to give radiolabeled precursors would not have been given, in the newborn and in pregnant women. The current use of stable isotopes in steroid research and endocrinology covers all branches of our field. In vivo metabolism of steroids is being studied, as are mechanistic aspects of metabolic transformations (e.g., aromatization). Determination of metabolic clearance rates and steroid quantification are important aspects of labeled steroid use. Finally, GUMS reference methodologies using stable isotopes have become the “gold standards” by which to verify im-
10.
I I.
12.
13.
14.
Schoenheimer R, Rittenberg D (1935). Deuterium as an indicator in the study of intermediary metabolism. Science 82:156-157. Urey HC, Brickwedde FG, Murphy GM (1932). A hydrogen isotope of mass 2. Physiol Rev 39164-165. Wood HG, Werkman CH, Hemingway A, Nier A0 (1940). Heavy carbon as a tracer in bacterial fixation of carbon dioxide. J Biol Chem 135~789-790. Taylor HS, Swingle WW, Eyring H, Frost AA (1933). The effect of water containing the isotope of hydrogen upon fresh water organisms. J Chem Physiol 1:75 1. Thompson JF (1963). Biological .Efecfs of Deurerium. Macmillan, London. Bloch K, Berg BN, Rittenberg D (1943). The biological conversion of cholesterol to cholic acid. J Biol Chem 149:511517. Bloch K (1945). The biological conversion of cholesterol to pregnanediol. J Biol Chem 157:661-X16. Bloch K. Borek E, Rittenberg D (1946). Synthesis of cholesterol in surviving liver. J Biol Chem 162:441-449. Bloch K (1952). Biological synthesis of cholesterol. Harvey Leer 48~68-88. Maudgal RK, Tchen TT, Bloch K (19.58). I ,2-Methyl shifts in the cyclization of squalene to lanosterol. J Biol Chem 80~2289-2590. Takes L, Throop LJ (1972). Introduction of deuterium into the steroid system. In: Fried J, Edwards JA (eds) Organic Reuctions in Steroid Chemistry. Van Nostrand Reinhold Co., New York, pp. 145-221. Braselton WE, Orr JC, Engel LL (1973). The twin-ion technique for detection of metabolites by gas chromatographymass spectrometry. Anal Biochem 53~64-85. Watkins JB, lngall D, Szczepanik P, Klein PD. Lester R (1973). Bile salt metabolism in the newborn. Measurement of pool size and synthesis by stable isotopic technique. J Med 28&43 I-434. Baillie TA, Anderson RA, Axelson M, Sjdvall K, Sjcvall J (1978). Pathways of steroid metabolism in uiuo studied by deuterium labelling techniques. In: Baillie TA (ed) Slab/e fsoropes. Macmillan, London, pp. 177-183.
Steroids,
1990, vol. 55, April
141
Coprostanone
Copfostanol
The need for precursor labeling had long been recognized and had been attempted using easily detectable adducts with benzene or halides, so the potential use of heavy isotopes was evident immediately after their discovery. This explains why only 3 years were to elapse between Urey’s discovery of deuterium’ and the first publication on the use of deuterium-IabeIed precursors in metabolic studies. The potential use of isotopes of other eIements present in biotogic systems was also recognized. Sulfur isotopes, i3C, isO, and lSN had all been isotated in the period from 1927 to 1930, and the first use of r3C in metabolism studies was that of Wood et al. ,? who demonstrated carbon dioxide fixation by bacteria grown in NaHi3C03 solutions. The i3C content of propionic, succinic, malic, and fumaric acids was determined using a “mass spectrograph.” it had been shown early that use of stable isotopelabeled compounds in intact organisms was probably safe. In 1933, Taylor et al.” had shown that fish and earthworms could survive in predominantly heavy water.* The early research on stable isotope use was spearheaded by Riftenb~rg and Schoenheimer at Co-
* For a later and more complete discussion of the toxicity of deuterium in mamm~s, the reader should consuit T~~mpson,s Address reprint requests to Dr. Cedric H. L. Shackleton, Children’s Hospital Oakland Research Institute, 747 2nd Street, Oakland, CA 94609. USA.
0 1990 Butterworth
Publishers
lumbia university, fater to be joined by Konrad Bfoch. Bloch and his coffeagues must be credited with leading steroid biosynthesis investigations during the 1940s and 1950s. An early example of steroid metabolic studies was their demonstration in 1943 that labeled cholic acid (a bile acid) was synthesized from labeled cholesterol by dogs.” In the first reported ~~~u~ expe~ment, Bfoch administered labeled cholesterol to a woman in the eighth month of pregnancy and isolated labeled pregnanedio1.7 These experiments showed for the first time that in the mammalian system, hormonal steroids and bile acids had choiesterof as a common precursor. If is remarkable that the first human experiment was on a pregnant woman; one senses that institutional review boards or their predecessors were non-existent in previous times. Of course, we now know that it is precisely in these sensitive situations (pregnancy or the newborn) that stable isotope labeling in metabolic studies should be encouraged for reasons of safety. One of the fnnd~ental early experiments in steroid biosynthesis was carried out by Bfoch and coworkers, who showed that cholesterof could be formed from deuterated or j3C-labeled acetate.g AII of these studies illustrate the fruitfulness of the period from 1935 to 1945 in biosynthetic studies using stable isotopes. Tracer methodology took a big Ieap after the war with the availability of i4C, which then became the focus for production of labeled precursors. For the next two decades, the use of 14C and 3H completely overshadowed the use of stable isotope-labeled materials. The decline of stable isotope work may be attributed to the unavailability of good reliable instrumentation for determination of isotope incorporation. A few mass spectrometers were available, but they were unreIiable and di~~ult to use and maintain. In contrast, by the late t9SOs, liquid scintillation counting had simplified detection of radioisotopes to the extent that novices could be readily trained in their use, In the haste to abandon stable isotope studies, it was frequently forgotten that the use of stable and radiolabeling techniques are complementary and that different information could be obtained. While radioactive measurement would tell you whether the product was Iabeled, it could not readily give info~ation on the position of the label. The CompIementary nature of the two techniques was weli-illustrated in the pioneering work on cholesterol synthesis from acetate via squalene carSteroids,
1990, vol. 55, April
139
I96Os, interest centered on studying biosynthetic pathways, which was most readily carried out with radiolabels. In contrast, studies performed during the 1970s and 1980s have focused to a greater extent on mechanistic aspects of biochemical t~nsfo~~tion~, in which information was required on the origin or f&e of’ specific atoms in a particular molecule. Interest in this field has been driven by pharmaceutical research, in which the conversion and breakdown of drugs must be accurately determined. Finally, one must mention the increased awareness of safety which has limited the widespread use of radiolabels in human metabolism studies. Investigations on the sequence of aromatization carried out by the late Lewis Engel and his group could be seen as introducing a new era of stable isotope studies. In a novel but simple experiment, they isolated several of the intermediates in placental microsomal estrogen formation.” By using as substrates testosterone or androstenedione, which were about 50% monodeuterated, all intermediates and the estrogen products were easily distinguishable by having M+ and [M + 11” of approximateiy equal size in their mass spectra. They termed this methodology the “twinion” technique. Figure i shows the spectra of isolated and reference estrone demonstrating the efficacy of this method. The current utilization of stable isotopes for in vivo steroid metabolism and turnover studies
ried out by Bloch.” By the judicious USC of acetate labeled in the one or two positions with ‘“C, or with a mixture of ‘“C and ‘IC, they were able to report in many cases which carbon atom of acetate gave rise to which carbon atom in choiestcrol. In their later experiment using squalene synthesized from 3-r3C acetoacetate and 4..rRC acetoacetate, they were able to determine exactly the mechanism of cyclization of squalene to lanosterol. The lanosterol had to be converted to ethylene for which the proportions of mono-‘%?, di“C, and unlabeled r3C could be determined.“’ The respective qualities of stable isotopes and radioisotopes in studies of metabolism have been summarized in Table 1. This table emphasizes the different roles that each plays, suggesting that with equal availability of suitable instrumentation for their measurement, both should be used. No single factor was responsible for the resurgence of interest in stable isotopes seen over the last few years. On one side was the increased availability of the isotopes themselves in high degrees of purity and an enlarging literature on how to introduce them into the steroid molecule.‘r and on another was the deveiopment and commercialization of mass spectrometers and nuclear magnetic resonance (NMR) instruments with which to detect them within biomolecules. The need for stable isotope studies was also widely recognized. With a few notable exceptions, in the 1950s and ftm 90.
iv+
73
342
\
BO-
COMPOUND
70-
ISOLATED
SO-
216 !
SOUOJO-
257 \
75 ./
327
10 0 low 90.
M' 342
73
BO-
0
70
uo-
MW
30.
342
244 232
I
M-28
i llll 60
60
100
120
I 1uo
II 160
II 180
II
III 200
I 220
II
‘8:
II 2uo
I 260
4
*St 280
.L 300
“,;:”
3j4
A
320
i
,.1. 31ia
ii.
360
M/E Figure 1 Mass spectra of trimethylsilyl ethers of estrone isolated from incubation of [7@+l]testosterone placental microsomes (top) and of the reference compound (bottom). (Reprinted with permission.lzl
140
Steroids,
1990, vol. 55, April
(42 atom % excess) with
Introduction: Table 1 Comparative features of stable and radioisotope topes in biochemical investigation Radiolabeled Can all common biologic elements be studied? Position of label in product easily discernable? Influence on substrate/product pool size Can total recovery be determined readily? Sensitivity of measurement Ease of measurement Use limited by ethical considerations? Suitability for use in enzyme mechanism studies Lower cost per experiment? Can be used for quantification? Abbreviations: tography-mass
No
iso-
Stable isotope-labeled
Shackleton
munoassays. In spite of greater availability of stable isotope-labeled steroids and better methods for determining their presence in compounds isolated from incubates or body fluids (e.g., NMR, high-pressure liquid chromatography-mass spectrometry, GUMS), their use remains severely limited. While stable isotopes are not appropriate for every investigation, I hope that the experimentation described in these pages will encourage more investigators to consider their use.
No
Yes (N and 0 have only stable isotope forms) Yes
None
Moderate
Yes
No
I.
High
2.
Easy Yes
Low (except for known metabolites) More difficult No
Low
High
4.
Yes
No
5.
Yes (RIA)
Yes (GUMS)
6.
References
RIA, radioimmunoassay; spectrometry.
GUMS,
gas chroma-
3.
I. 8. 9.
can be traced to the bile acid studies of Watkins et aLi and the steroid sulfate metabolism studies by Baillie et aLi Both of these investigations were carried out in situations in which permission to give radiolabeled precursors would not have been given, in the newborn and in pregnant women. The current use of stable isotopes in steroid research and endocrinology covers all branches of our field. In vivo metabolism of steroids is being studied, as are mechanistic aspects of metabolic transformations (e.g., aromatization). Determination of metabolic clearance rates and steroid quantification are important aspects of labeled steroid use. Finally, GUMS reference methodologies using stable isotopes have become the “gold standards” by which to verify im-
10.
I I.
12.
13.
14.
Schoenheimer R, Rittenberg D (1935). Deuterium as an indicator in the study of intermediary metabolism. Science 82:156-157. Urey HC, Brickwedde FG, Murphy GM (1932). A hydrogen isotope of mass 2. Physiol Rev 39164-165. Wood HG, Werkman CH, Hemingway A, Nier A0 (1940). Heavy carbon as a tracer in bacterial fixation of carbon dioxide. J Biol Chem 135~789-790. Taylor HS, Swingle WW, Eyring H, Frost AA (1933). The effect of water containing the isotope of hydrogen upon fresh water organisms. J Chem Physiol 1:75 1. Thompson JF (1963). Biological .Efecfs of Deurerium. Macmillan, London. Bloch K, Berg BN, Rittenberg D (1943). The biological conversion of cholesterol to cholic acid. J Biol Chem 149:511517. Bloch K (1945). The biological conversion of cholesterol to pregnanediol. J Biol Chem 157:661-X16. Bloch K. Borek E, Rittenberg D (1946). Synthesis of cholesterol in surviving liver. J Biol Chem 162:441-449. Bloch K (1952). Biological synthesis of cholesterol. Harvey Leer 48~68-88. Maudgal RK, Tchen TT, Bloch K (19.58). I ,2-Methyl shifts in the cyclization of squalene to lanosterol. J Biol Chem 80~2289-2590. Takes L, Throop LJ (1972). Introduction of deuterium into the steroid system. In: Fried J, Edwards JA (eds) Organic Reuctions in Steroid Chemistry. Van Nostrand Reinhold Co., New York, pp. 145-221. Braselton WE, Orr JC, Engel LL (1973). The twin-ion technique for detection of metabolites by gas chromatographymass spectrometry. Anal Biochem 53~64-85. Watkins JB, lngall D, Szczepanik P, Klein PD. Lester R (1973). Bile salt metabolism in the newborn. Measurement of pool size and synthesis by stable isotopic technique. J Med 28&43 I-434. Baillie TA, Anderson RA, Axelson M, Sjdvall K, Sjcvall J (1978). Pathways of steroid metabolism in uiuo studied by deuterium labelling techniques. In: Baillie TA (ed) Slab/e fsoropes. Macmillan, London, pp. 177-183.
Steroids,
1990, vol. 55, April
141
