Table Of ContentSteroid
Biochemistry
Volume II
Editor
Ronald Hobkirk, Ph.D., D. Sc.
Professor of Biochemistry
University of Western Ontario
London, Ontario, Canada
Boca Raton London New York
CRC PressC isR anC im Pprrinets osf, thIenc.
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Steroid biochemistry.
Includes index.
1. Steroid hormone metabolism. 2. Biological
chemistry. I. Hobkirk, Ronald, 1930- [DNLM:
1. Steroids — Biosynthesis. 2. Steroids — Metabolism.
QU85 S8355]
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ISBN 0-8493-5194-4 (v. 2)
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PREFACE
It was initially anticipated that about eight or nine chapters on various topics in
steroid biochemistry would constitute a single volume of this publication. However, it
became apparent that the literature in certain of the areas covered was so considerable
as to necessitate two volumes, each containing four chapters. Since it was clearly ad-
vantageous that these volumes should be of approximately equal length, the original
order of chapters had to be altered somewhat. This change, however, has not resulted
in any major disadvantage.
An attempt has been made to attract contributions which illustrate the importance
of certain enzymatic processes involved in steroid biosynthesis and metabolism and,
in some cases, leading to steroidal action in target sites. Investigators actively engaged
in research in such areas were invited to present their material in a manner which they
considered fitting. It is hoped that as a result of this, the publication will possess suf-
ficient depth to warrant approval. The blend of review material and experimental data
originating in the authors' laboratories will, it is felt, make for useful reading.
As is the case with virtually every publication which involves multiple contributors,
the various chapters were completed at rather different times, resulting in some being
more current than others. For this reason dates of receipt are included in the following
brief description of the contents.
In Volume I the chapter by A. F. Clark (July 1978) deals with the reductase enzymes
acting at carbon-5 of the steroid molecule, with particular emphasis upon the potential
hormonal activities of the products. An in-depth view of the chemistry and biochem-
istry of steroid carboxylic acids is provided by H. L. Bradlow and C. Monder (Novem-
ber 1977). It should be noted that the original work of these authors provided most of
our current knowledge on this topic. D. G. Williamson's review (June 1977) of the
biochemistry of 17 -hydroxysteroid dehydrogenases focuses upon the role of these en-
zymes in the biological activation and inactivation of steroids. A. H. Payne and S. S.
Singer (July 1977) discuss the role of steroid sulfotransferase and sulfatase systems
from a biochemical viewpoint. In addition, they draw attention to the possible biolog-
ical role of glucocorticoid sulfates. Volume II of the publication contains a chapter by
B. R. Bhavnani and C. A. Woolever (August 1977) on biosynthetic pathways and some
aspects of metabolism and biological activity of ring B unsaturated estrogens. This is
a topic which has been primarily elucidated by the experimental work of Bhavnani. J-
G. Lehoux (July 1977) reviews the complex area of the control of mineralocorticoid
biosynthesis and the involvement of peptide hormones, electrolytes, prostaglandins,
serotonin, etc. P. I. Musey, K. Wright, J. R. K. Preedy and D. C. Collins (July 1978)
deal with steroid conjugate formation in relation to steroid distribution in body tissues
and fluids. Factors related to the biological hydroxylation of 18- and 19-carbon ste-
roids are discussed in a chapter by R. Hobkirk (May 1977).
It is my distinct pleasure to thank each contributor for the effort put into this pub-
lication. I should also like to acknowledge the help accorded me by CRC Press, partic-
ularly by Coordinating Editor Benita Segraves.
R. Hobkirk
London, Canada
THE EDITOR
Dr. R. Hobkirk was born in the town of Peebles in Scotland and attended the local
High School. After graduating B.Sc. with Honors in Biochemistry from the University
of Edinburgh in 1952 he completed a Ph.D. degree at the same institution in 1955.
This latter involved studies on the biochemistry of plant cell wall polysaccharides. The
following two years were spent as a research fellow (British Empire Cancer Campaign)
in the Departments of Biochemistry and Surgery, University of Glasgow. During that
period studies were performed on the relationship of steroid hormones to human breast
cancer. Part of the 1957-'58 year was spent as a research fellow in the Department
of Biochemistry, McGill University, Montreal and then, until 1960, in the Department
of Metabolism, The Montreal General Hospital, Montreal. In 1960 Dr. Hobkirk was
appointed Assistant Professor in Experimental Medicine, McGill University, followed
in 1962 by promotion to Associate Professor and in 1967 to Professor (Biochemistry)
in the Department of Medicine. In 1966 he was appointed a Research Associate of the
Medical Research Council of Canada (i.e., a full time investigatorship), an award
which he continues to hold. In 1971 he moved to the University of Western Ontario,
London, Canada, and is currently Professor in the Department of Biochemistry. He
is occupied with research and teaching in hormone biochemistry. In 1973 he received
the degree of D.Sc. from the University of Edinburgh for a thesis entitled, "Metabo-
lism of the Estrogens and their Conjugates".
CONTRIBUTORS
Bhagu R. Bhavnani, Ph.D. Paul I. Musey, Ph.D.
Associate Professor Assistant Professor of Medicine
Department of Obstetrics and Emory University School of Medicine
Gynecology Atlanta, Georgia
McMaster University
Hamilton, Ontario, Canada Anita H. Payne, Ph.D.
Associate Professor of Biological
H. Leon Bradlow, Ph.D. Chemistry
The Rockefeller University Department of Obstetrics and Gynecol·
New York City, New York ogy
University of Michigan
Albert F. Clark, Ph.D. Ann Arbor, Michigan
Professor of Biochemistry
and Associate Professor of Pathology John R. K. Preedy, M.D.
Queen's University Professor of Medicine,
Kingston, Ontario, Canada Associate Professor of Biochemistry
Emory University School of Medicine
Delwood C. Collins, Ph.D. Atlanta, Georgia
Professor of Medicine
Associate Professor of Biochemistry Sanford S. Singer, Ph.D.
Emory University School of Medicine Associate Professor of Chemistry
Atlanta, Georgia University of Dayton
Dayton, Ohio
Ronald Hobkirk, Ph.D., D. Sc.
Professor of Biochemistry Denis G. Williamson, Ph.D.
University of Western Ontario Associate Professor
London, Ontario, Canada Department of Biochemistry
University of Ottawa
Jean-Guy Lehoux, Ph.D. Ottawa, Ontario, Canada
Associate Professor, Faculty of
Medicine Charles A. Woolever, M.D.
Sherbrooke University Professor of Obstetrics and
Sherbrooke, Quebec, Canada Gynecology
McMaster University
Carl Monder, Ph.D. Hamilton, Ontario, Canada
Professor of Biochemistry
Mt. Sinai School of Medicine Kristina Wright, Ph.D.
Director, Section on Steroid Studies Instructor of Medicine
Hospital for Joint Diseases Emory University School of Medicine
New York, New York Atlanta, Georgia
TABLE OF CONTENTS
Volume I
Chapter 1
Steroid A4 Reductases: Their Physiological Role and Significance ................. 1
Albert F. Clark
Chapter 2
Steroid Carboxylic Acids .................................................. 29
H. L. Bradlow and C. Monder
Chapter 3
Biochemistry of Steroid 17-Hydroxysteroid Dehydrogenases .................... 83
D. G. Williamson
Chapter4
The Role of Steroid Sulfatase and Sulfotransferase Enzymes in the Metabolism of C21
c.,
and Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
A. H. Payne and S. S. Singer
Index ................................................................. 147
Volume II
Chapter 1
Alternative Pathways of Steroid Biosynthesis and the Origin, Metabolism, and
Biological Effects of Ring B Unsaturated Estrogens ............................ 1
B. R. Bhavnani and C. A. Woolever
Chapter2
Factors Controlling the Biosynthesis of Aldosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
J.-0. Lehoux
Chapter 3
Influence of Steroid Conjugation on Excretion and Tissue Distribution ............ 81
P. I. Musey, K. Wright, J. R. K. Preedy, and D. C. Collins
Chapter4
c.,
Hydroxylated C18 and Steroids: Their Significance and Factors Related to Their
Biosynthesis ............................................................ 133
R. Hobkirk
Index .................................................................. 181
Chapter 1
ALTERNATE PATHWAYS OF STEROID BIOSYNTHESIS AND THE
ORIGIN, METABOLISM, AND BIOLOGICAL EFFECTS OF RING B
UNSATURATED ESTROGENS
B. R. Bhavnani and C. A. Woolever
TABLE OF CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
II. Classical Pathway of Steroidogenesis ................................... 2
A. Source of Carbon Atoms of Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . 2
B. Biosynthesis of Mevalonic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
C. Formation of Isopentenyl Pyrophosphate from Mevalonic Acid . . . . . . 3
D. Formation of Squalene from lsopentenyl Pyrophosphate ............ 4
E. Cyclization of Squalene to Lanosterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
F. Conversion of Lanosterol to Cholesterol. ......................... 7
G. Conversion of Cholesterol to Pregnenolone ....................... 7
III. Alternate Pathways of Steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
A. Direct Conversion of Desmosterol to Steroid Hormones ............. 9
B. Sesterterpene Pathway of Steroidogenesis ......................... 10
C. Biosynthesis of Ring B Unsaturated Estrogens ..................... 11
1. Metabolism of 7-3H-Dehydroepiandrosterone and 4-14C-Andros-
tenedione Injected into the Umbilical Vein .................. 15
2. Metabolism of 1-14C-Sodium Acetate and 7-3H-Cholesterol In-
jected into the Fetal Circulation ........................... 17
3. Metabolism of 14C-Squalene and 14C-Mevalonic Acid Injected
into the Umbilical Circulation ............................. 20
4. Metabolism of 1-14C-Isopentenyl Pyrophosphate and (4,8,12)-
14C-Farnesyl Pyrophosphate Injected into the Fetus .......... 21
5. Theoretical Discussion on Alternate Pathways of Steroido-
genesis ............................................... 24
IV. Biologic Activity of Ring B Unsaturated Estrogens ....................... 25
V. Bioassays .......................................................... 26
A. General ..................................................... 26
B. Animal Bioassay Studies ....................................... 27
1. Allen-Doisy Test ........................................ 27
2. Uterotrophic Assay ..................................... 28
3. Chick Oviduct Assay .................................... 29
C. Human Studies ............................................... 30
1. Specific Responses ...................................... 30
2. Other Responses in the Human ............................ 32
a. Hypothalamus and Pituitary ........................ 32
b. Clotting Mechanism ............................... 32
c. Metabolic Changes ................................ 32
d. Osteoporosis ..................................... 33
2 Steroid Biochemistry
e. Atherosclerosis ................................... 33
f. Neoplasms of the Reproductive Organs ............... 34
D. The Metabolism of Equilin in Man ............................... 35
1. Intravenous Equilin Sulfate ............................... 35
2. Intravenous 3H-Equilin .................................. 36
VI. Concluding Remarks ................................................ 42
Acknowledgments ........................................................ 43
References ............................................................... 43
I. INTRODUCTION
For the past 25 years, it has been generally accepted that cholesterol is the obligatory
precursor in the biosynthesis of all steroid hormones. Details of the various steps for
the synthesis of cholesterol from acetate and for its conversion to steroids were estab-
lished mainly by in vitro incubation procedures using tissues such as the ovary, testis,
and adrenal cortex. Although it became firmly established that acetate was the initial
two carbon source of cholesterol, some experimentation suggested that cholesterol may
not be the only precursor from which all steroid hormones are formed.
This chapter will discuss the evidence for the steroidogenic pathway which bypasses
cholesterol. It will be referred to as the alternate pathway of steroidogenesis, while
that which utilizes cholesterol as an obligatory intermediate will be termed the classical
pathway of steroidogenesis. Although it is not the purpose of this chapter to discuss
the classical pathway of biosynthesis and metabolism of steroid hormones, ·its salient
features will be reviewed briefly to facilitate comparison with the alternate pathway,
which will be discussed in detail. The metabolism and biological action of the ring B
unsaturated estrogens will also be presented.
II. CLASSICAL PATHWAY OF STEROIDOGENESIS
The various steps in the classical pathway of steroidogenesis from acetate are acetate
-+ mevalonic acid -+ isopentenyl pyrophosphate -+ 3,3-dimethyl allyl pyrophosphate
-+ geranyl pyrophosphate -+ farnesyl pyrophosphate -+ squalene -+ lanosterol __.. cho-
lesterol-+ pregnenolone -+ steroid hormones. The details are discussed below.
A. Source of Carbon Atoms of Cholesterol
Studies by Bloch, Lynen, Cornforth, Popjak, and their colleagues on cholesterol
biosynthesis from either methyl or carboxyl 14C-labeled acetate established that all 27
carbon atoms of cholesterol are derived from acetate. These investigations have been
reviewed by Bloch. 1 The distribution of the 15 methyl and 12 carboxyl carbons in
cholesterol is shown in Figure 1. This arrangement of the carbon atoms of acetate in
cholesterol follows the "isoprene rule," which was defined by Ruzicka as a working
hypothesis as early as 1921.2
B. Biosynthesis of Mevalonic Acid
Three molecules of acetic acid in the form of acetyl-CoA (coenzyme A) thioester
3
FIGURE I. Distribution of carboxyl(C) and methyl(M)
carbons of acetic acid in cholesterol.
0 0 0
11 fi-KETOTHIOLASE II II
2CH 3 -C-5-CoA CH rC-CH2-C-S-CoA Co-ASH
ACETYl-GoA ACETOACETYl-GoA COENZYME A
+ Co-ASH
3-HYDROXY-3-METHYL GLUTARYL-CoA
MEVALONATE-NADP OXIDO ~ 2 NADPH + 2H+
REDUCTASE EC 111 34 ~ 2 NADP+
H3C /OH
OJ=C' c"{'-< ;-"' -cH( C'H-'2o "
3R'-MEVALONIC ACID
FIGURE 2. Biosynthesis of mevalonic acid from acetyl
coenzyme A.
condense to form mevalonic acid (Figure 2). All of the intermediates involved are
bound to coenzyme A, and the required enzymes are present in both the cytoplasmic
particles and the soluble fraction of mammalian and avian liver3 6 and in yeast. 78 The
- '
first step, catalyzed by a soluble enzyme (3-keto-thiolase, results in the formation of
acetoacetyl-CoA. In the presence of 3-hydroxy-3-methyl-glutaryl-CoA synthase (which
is located in the mitochondria and the soluble fraction of liveruo but not in the micro-
somal fraction as had been reported previously'u2 the acetoacetyl-CoA condenses
),
with a third molecule of acetyl-CoA to give 3-hydroxy-3-methyl-glutaryl-CoA (HMG-
CoA), the immediate precursor of mevalonic acid. The reduction of this precursor to
mevalonic acid is catalyzed by mevalonate: NADP• oxidoreductase (EC 1.1.1.34),
which is present in both yeast mitochondria 13 and in the mammalian liver micro-
somes. 'us This microsomal system is thought to be the rate-limiting step in the biosyn-
thesis of cholesterol in mammalian liver.1u 6 17 Although this scheme is generally be-
,
lieved to be the major pathway of mevalonate biosynthesis, an alternate minor
pathway involving malonyl-CoA has been described by Brodie et al. '8 9 The mecha-
·'
nisms involved in these transformations have been recently reviewed by Beytia and
Porter.20
C. Formation of lsopentenyl Pyrophosphate From Mevalonic Acid
The biosynthesis of isopentenyl pyrophosphate (IPP) from 3R-mevalonic acid is cat-
alyzed by soluble enzymes and involves phosphorylated intermediates. Mevalonic acid
is sequentially phosphorylated first (Figure 3) with ATP in the presence of mevalonic
