A guide to understanding the steroid pathway: New insights and diagnostic implications

doi:10.1016/j.clinbiochem.2014.07.017

Clinical Biochemistry

Volume 47, Issue 15, October 2014, Pages 5-15

https://doi.org/10.1016/j.clinbiochem.2014.07.017 Get rights and content

Highlights

•
Understanding the steroid pathway provides clarity for interpretation of complex patient results
•
Serum and urine metabolomic profiles are useful to investigate disorders of sex development
•
Interpretation of steroid results in the neonate remains challenging
•
Foetal adrenal enzyme function persists, at least to 40 weeks corrected gestation age
•
The alternative steroid pathway has potential implications for human sex development

Abstract

Steroid analysis has always been complicated requiring a clear understanding of both the clinical and analytical aspects in order to accurately interpret results. The literature relating to this specialised area spans many decades and the intricacies of the steroid pathway have evolved with time. A number of key changes, including discovery of the alternative androgen pathway, have occurred in the last decade, potentially changing our understanding and approach to investigating disorders of sexual development. Such investigation usually occurs in specialised paediatric centres and although preterm infants represent only a small percentage of the patient population, consideration of the persistence of the foetal adrenal zone is an additional important consideration when undertaking steroid hormone investigations. The recent expanded role of mass spectrometry and molecular diagnostic methods provides significant improvements for accurate steroid quantification and identification of enzyme deficiencies. However analysis of steroids and interpretation of results remain complicated. This review aims to provide an insight into the complexities of steroid measurement in children and offers an updated guide to interpretation, of serum and urine steroids through the presentation of a refined steroid pathway.

Graphical abstract

Introduction

Since the development of the early radioimmunoassays (RIA) for serum steroids [1] and gas chromatography (GC) metabolomic methods for urine steroids [2], [3], [4], [5] in the 1960's, there have been significant advances in our understanding and quantification of these remarkable hormones. Recent refinements in mass spectrometry and molecular techniques have resulted in a paradigm shift in steroid analysis. Appreciation of the advantages and limitations of the analytical methods aids clinical interpretation. Even so, due to the number and similarity of many steroids the interpretation of results remains complicated; particularly in neonates. A clear understanding of steroid structure, the genetic basis of steroid biosynthesis and the relationship between steroids in the pathway is essential for accurate interpretation. With the recognition of the newly discovered alternative androgen pathway and the advent of mass spectrometry in the clinical diagnostic laboratory, it is timely to examine the current advances in steroid biosynthesis and analysis.

This review aims to provide an insight into the complexities of steroid measurement in children and offers an updated guide to the interpretation of serum and urine steroids through the presentation of a refined steroid pathway.

Section snippets

Structure and naming of steroids

Historically many of the current trivial steroid hormone names owe their origin to scientific endeavours of the early 20th century[6], [7]. The isolation and naming of the first steroids related to their broad function, such as the names coined for “estrone” (estr(us)= fertile female; and one = ketone), “androsterone” (andro = male; ster = sterol; and one = ketone) and “testosterone” (testo = testes; ster = sterol; and one = ketone)[8]. As the repertoire of steroids of clinical interest expanded individual

Steroid synthesis

All steroids are synthesised from cholesterol. Tissues that can convert cholesterol with the cytochrome P450 side chain cleavage (P450scc) enzyme have the ability to produce steroids. In humans several organs are capable of steroidogenesis: adrenal cortex (zona glomerulosa, zona fasciculata and zona reticularis); Leydig cells of the testes; granulosa and theca cells of the ovary; and syncytiotrophoblasts of the placenta. It is also possible that some steroidogenesis takes place in the human

Steroid synthesis and metabolism in the foetus and neonate

The process of steroid production and metabolism described so far relates to children and adults. Both gender and age influence steroid production. The most significant changes in steroid levels and pattern occur during the neonatal period, at adrenarche, puberty, pregnancy and also menopause. Of these milestones, interpretation of steroids in the neonate remains the most challenging. To understand some of the complications with interpretation in the neonate, a background understanding of the

The alternative steroid pathway

The “alternative” or “backdoor” sex steroid pathway, identified in 2003 in the pouch young of the Australian Tammar wallaby, is now recognised to have potential implications for human sex development[35]. Formation of dihydrotestosterone (DHT) is central to both the classical and alternative androgen pathways. In humans, the classical pathway to formation of DHT involves its conversion from testosterone by the enzyme 5α-reductase, whereas this mechanism is bypassed in the alternative pathway.

Laboratory investigation of steroid biosynthesis

For the clinical scientist the fundamental basis for interpreting steroid results relies on a clear appreciation of the analytical method, the structure of related steroids, their relationship in the steroid pathway and clear “normal” ranges. However analysis of steroids and interpretation of results has remained complicated due to the number and similarity of steroids, plus the matrices in which steroids are measured. In addition, analytical performance characteristics, such as specificity,

Normal physiological results

Steroid levels and the pattern observed in serum and urine samples change with age. These changes mark key events in maturity and are most marked in the neonatal period, onset of adrenarche and puberty.

Conclusion

Laboratory techniques for steroid analysis have progressed significantly over the last twenty plus years. In an already complicated area, the interpretation of steroid results potentially is now more challenging with our new understanding of the steroid cascade, its clinical relevance to DSD, advances in laboratory technology and interpretation of steroid results. The future brings a combination of next generation sequencing (genomics) in association with mass spectrometry (metabolomics) to

Funding

In house funding from the Murdoch Children's Research Institute was provided for the graphic artists' work associated with Fig. 2, Fig. 3. There was no other source of funding related to this work.

Financial disclosure

The authors have no financial relationships relevant to this article to disclose.

Conflict of interest

All authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this manuscript.

Contributor statement/disclosure summary

There is no conflict of interest that could be perceived as prejudicing the impartiality of this manuscript. All authors listed contributed to this work. Dr Greaves wrote the first draft of this manuscript and designed the first version of the steroid pathway. Dr Jevalikar performed the background search for the work in Table 2, Table 3 and together with Dr Hewitt and Prof Zacharin contributed to the refinement of the steroid pathway presented in this manuscript. Professor Zacharin critically

Acknowledgements

We thank Mr Bill Reid for his patience and attention to detail in relation to the production of Fig. 2, Fig. 3.

References (95)

E.C. Horning et al.
Gas phase analytical methods for the study of steroids
Adv Lipid Res
(1968)
C. Shackleton
Clinical steroid mass spectrometry: a 45-year history culminating in HPLC–MS/MS becoming an essential tool for patient diagnosis
J Steroid Biochem Mol Biol
(2010)
F.L. Mitchell et al.
The investigation of steroid metabolism in early infancy
Adv Clin Chem
(1969)
E.R. Freeman et al.
A brief history of testosterone
J Urol
(2001)
O. Wintersteiner et al.
Chemical studies on the adrenal cortex
J Biol Chem
(1936)
T. Reichstein et al.
The hormones of the adrenal cortex
Vitam Horm
(1943)
V. Luu-The
Assessment of steroidogenesis and steroidogenic enzyme functions
J Steroid Biochem Mol Biol
(2013)
R. Andrew
Clinical measurement of steroid metabolism
Best Pract Res Clin Endocrinol Metab
(2001)
R. Auchus et al.
46, XX DSD: the masculinised female
Best Pract Res Clin Endocrinol Metab
(2010)
R.J. Auchus
The alternative pathway to dihydrotestosterone
Trends Endocrinol Metab
(2004)

C.E. Flück et al.

Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual development

Am J Hum Genet

(2011)

C. Rossi et al.

Serum steroid profiling for congenital adrenal hyperplasia using liquid chromatography–tandem mass spectrometry

Clin Chim Acta

(2010)

G. Abraham et al.

Combined radioimmunoassay of four steroids in one ml plasma: II. Androgens

Clin Biochem

(1975)

C. Wang et al.

Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: interference and comparison with established methods

Steroids

(2008)

H. Licea-Perez et al.

Development of a highly sensitive and selective UPLC/MS/MS method for the simultaneous determination of testosterone and 5-dihydrotestosterone in human serum to support testosterone replacement therapy for hypogonadism

Steroids

(2008)

L. Starka

Epitestosterone

J Steroid Biochem Mol Biol

(2003)

R.F. Greaves et al.

Serum Hormone Profiles over the first six weeks of life for preterm infants born less than 30 weeks gestation

R.F. Greaves et al.

Establishment of hormone reference intervals for infants born < 30 weeks' gestation

Clin Biochem

(2014)

R.F. Greaves

Hormone profiles in extremely preterm infants

Clin Biochem

(2014)

R.C. Wilson et al.

Ethnic Specific Distribution of Mutations in 716 Patients with Congenital Adrenal Hyperplasia Owing to 21-Hydroxylase Deficiency

Mol Genet Metab

(2007)

S. Christakoudi et al.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 4. Characterization, using GC–MS and GC–MS/MS, of 11oxo-pregnanes and 11oxo-pregnenes

Steroids

(2013)

G.E. Abraham

Solid-phase radioimmunoassay of estradiol-17 beta

J Clin Endocrinol Metab

(1969)

C.C. Sweeley et al.

Microanalytical separation of steroids by gas chromatography

Nature

(1960)

E.H. Starling

The Croonian lectures on the correlation of the functions of the body

Lancet

(1905)

G. Liljestrand

The Nobel Prize in physiology or medicine 1950: award ceremony speech

E.C. Kendall

The development of cortisone as a therapeutic agent

Nobel lecture, December 11, 1950

(1950)

T. Reichstein

Chemistry of the adrenal cortex hormones

P.L. Julian et al.

Sterols. XI. 17α-hydroxy-11-desoxycorticosterone (Reichstein's substance S)

J Am Chem Soc

(1950)

IUPAC-IUB Joint commission on biochemical nomenclature (JCBN)

The nomenclature of steroids

Eur J Biochem

(1989)

IUPAC-IUB Joint commission on biochemical nomenclature (JCBN)

The nomenclature of steroids

Pure Appl Chem

(1989)

IUPAC-IUB Joint commission on biochemical nomenclature (JCBN)

The nomenclature of steroids

R. Haining

Cytochrome P450 reactions in the human brain

J.F. Griffin et al.

Atlas of steroid structure volume 2: published online in 2010

Biochemical education

(2010)

R.M. Carey

Adrenal disease update 2011

J Clin Endocrinol Metab

(2011)

R. Greaves et al.

Genital abnormalities mimicking congenital adrenal hyperplasia in premature infants

J Paediatr Child Health

(2004)

R. Greaves et al.

11β-hydroxylase deficiency masked by alternative medicines

J Paediatr Child Health

(2006)

J.W. Honour

Steroid profiling

Ann Clin Biochem

(1997)

S.A. Wudy et al.

Mass spectrometry in the diagnosis of steroid-related disorders: clinical applications

K.M. McNamara et al.

Phase two steroid metabolism and its roles in breast and prostate cancer patients

Front Endocrinol

(2013)

C.H.L. Shackleton

Steroid synthesis and catabolism in the fetus and neonate

P.C. Midgley et al.

Plasma cortisol, cortisone and urinary glucocorticoid metabolites in preterm infants

Biol Neonate

(2001)

I. Vermes et al.

Maturation of the circadian rhythm of the adrenocortical functions in human neonates and infants

Horm Res

(1980)

P.C. Midgley et al.

Activity of the adrenal fetal zone in preterm infants continues to term

Endocr Res

(1996)

W.E. Rainey et al.

The human fetal adrenal: making adrenal androgens for placental estrogens

Semin Reprod Med

(2004)

R. Greaves et al.

Transient anomalies in genital appearance in some extremely preterm female infants may be the result of foetal programming causing a surge in LH and the over activation of the pituitary-gonadal axis

Clin Endocrinol (Oxf)

(2008)

J.D. Wilson et al.

5α-androstane-3α, 17β-diol is formed in tammar wallaby pouch young testes by a pathway involving 5α-prenane-3α, 17α-diol-20-one as a key intermediate

Endocrinology

(2003)

A.M. Biogiovanni

Urinary pregnanetriol. A practical determination in clinical medicine

Clin Endocrinol (Oxf)

(1960)

Cited by (35)

Associations of maternal exposure to 2,4-dichlorophenoxyacetic acid during early pregnancy with steroid hormones among one-month-old infants
2024, Science of the Total Environment
Exposure to 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used hormonal herbicide, may disrupt steroid hormone homeostasis. However, evidence from population-based studies is limited, especially for one-month-old infants whose steroid hormones are in a state of adjustment to extrauterine life and can be important indicators of endocrine development. This study aimed to explore the associations between maternal 2,4-D exposure during early pregnancy and infant steroid hormone levels.
The 885 mother-infant pairs were from a birth cohort in Wuhan, China. Maternal exposure to 2,4-D was determined in urine samples from early pregnancy, and nine steroid hormones were determined in infant urine. The associations of maternal 2,4-D exposure with infant steroid hormones and their product-to-precursor ratios were estimated based on generalized linear models, and bioinformatic analysis was conducted with public databases to explore the potential mechanisms involved.
The detection frequency of 2,4-D was 99.32 %, and the detection frequency of steroid hormones ranged from 98.42 % to 100.00 %. After adjusting for covariates, an interquartile range increase in 2,4-D concentrations was associated with a 7.84 % decrease in 11-deoxycortisol (95 % confidence interval, CI: −14.12 %, −1.10 %), an 8.09 % decrease in corticosterone (95 % CI: −14.56 %, −1.14 %), an 8.67 % decrease in cortisol (95 % CI: −14.43 %, −2.52 %), a 13.00 % decrease in cortisone (95 % CI: −20.64 %, −4.62 %), and an 11.17 % decrease in aldosterone (95 % CI: −19.62 %, −1.83 %). Maternal 2,4-D was also associated with lower infant cortisol/17α-OH-progesterone, cortisol/pregnenolone, and aldosterone/pregnenolone ratios. In bioinformatic analysis, pathways/biological processes related to steroid hormone synthesis and secretion were enriched from target genes of 2,4-D exposure.
Maternal urinary 2,4-D during early pregnancy was associated with lower infant urinary 11-deoxycortisol, corticosterone, cortisol, cortisone, and aldosterone, reflecting that 2,4-D exposure may interfere with infant steroid hormone homeostasis. Further efforts are still needed to study the relevant health effects of exposure to 2,4-D, particularly for vulnerable populations.
Cortisol in deciduous tooth tissues: A potential metric for assessing stress exposure in archaeological and living populations
2023, International Journal of Paleopathology
Cortisol is a glucocorticoid hormone produced by the hypothalamic-pituitary-adrenal axis that is regularly assessed in modern human and non-human populations in saliva, blood, and hair as a measure of stress exposure and stress reactivity. While recent research has detected cortisol concentrations in modern and archaeological permanent dental tissues, the present study assessed human primary (deciduous) teeth for cortisol concentrations.
Fifty-one dentine and enamel samples from nine modern and 10 archaeological deciduous teeth were analyzed for cortisol concentrations via enzyme-linked immunosorbent assay (ELISA).
Detectable concentrations of cortisol were identified in 15 (of 32) dentine and 8 (of 19) enamel samples coming from modern and archaeological deciduous teeth.
This study is the first known analysis of cortisol from deciduous dental tissues, demonstrating the potential to identify measurable concentrations.
The ability to analyze deciduous teeth is integral to developing dental cortisol methods with multiple potential future applications, including research on the biological embedding of stress in the skeleton. This study marks a key step in a larger research program to study stress in primary dentition from living and archaeological populations.
Multiple samples generated cortisol values that were not detectable with ELISA. Minimum quantities of tissue may be required to generate detectable levels of cortisol.
Future research should include larger sample sizes and consideration of intrinsic biological and extrinsic preservation factors on dental cortisol. Further method validation and alternative methods for assessing dental cortisol are needed.
Sex steroid metabolism and action in colon health and disease
2023, Journal of Steroid Biochemistry and Molecular Biology
The colon is the largest hormonally active tissue in the human body. It has been known for over a hundred years that various hormones and bioactive peptides play important roles in colon function. More recently there is a growing interest in the role the sex steroids, oestrogens and androgens, may play in both normal colon physiology and colon pathophysiology. In this review, we examine the potential role oestrogens and androgens play in the colon. The metabolism and subsequent action of sex steroids in colonic tissue is discussed and how these hormones impact colon motility is investigated. Furthermore, we also determine how oestrogens and androgens influence colorectal cancer incidence and development and highlight potential new therapeutic targets for this malignancy. This review also examines how sex steroids potentially impact the severity and progression of other colon disease, such as diverticulitis, irritable bowel syndrome, and polyp formation.
Cholesterol metabolism is decreased in patients with diminished ovarian reserve
2022, Reproductive BioMedicine Online
Citation Excerpt :
These results imply that aberrant regulation of SREBF/SCAP may decrease cholesterol synthesis and transport in granulosa cells. Cholesterol functions as a precursor molecule in the synthesis of steroid hormones (Greaves et al., 2014). The present study found that expression of the steroidogenic mediator CYP19A1 was significantly decreased in the DOR group (Figure 2).
Does cholesterol metabolism differ in patients with diminished ovarian reserve (DOR) compared to patients with normal ovarian reserve (NOR)?
The current research included 72 women with NOR and 86 women with DOR. Data on the cholesterol metabolism in granulosa cells of these women were analysed.
On the day of human chorionic gonadotrophin injection, serum oestradiol and progesterone in the DOR group were significantly lower than in the control group (P < 0.001). There were no significant differences in serum concentrations of total cholesterol, triglyceride, high-density lipoprotein and low-density lipoprotein between the NOR and DOR groups. The cholesterol-regulated gene SCAP in granulosa cells from women with DOR was down-regulated (P = 0.024). Cholesterol synthesis and transport genes (e.g. IDI1, FDFT1, CYP51A1, SRB1 and STARD1) were also significantly decreased (P = 0.026, P = 0.044, P = 0.049, P = 0.004 and P < 0.001, respectively). In granulosa cells of patients with DOR, cholesterol-related substances such as coprostanone, 11A-acetoxyprogesterone and 17α-hydroxyprogesterone were significantly reduced (P = 0.0008, P = 0.0269, P = 0.0337, respectively). CYP19A1, a key steroidogenesis gene, was significantly reduced (P = 0.009). 17α-hydroxyprogesterone and oestradiol decreased (P = 0.004 and P = 0.039, respectively).
Decreased cholesterol metabolism affecting steroid hormone synthesis in granulosa cells might be a possible mechanism for DOR.
A study on steroidogenic elaborations of stroma and their regulation in response to ovarian hormones in goats
2021, Animal Reproduction Science
Stromal tissue is an essential componenlt of the ovary not only for providing structural support but also for contributing to the early follicular growth with their bi-directional paracrine signaling. Estradiol is a major female hormone mainly secreted by the follicular cells in the ovary. To examine the relationship between 17β-estradiol and the factors involved in androgen production in stromal cells, ovarian stromal cells were cultured in the graded concentrations (50 and 100 ng/mL) of 17β-estradiol for varying time periods (24 and 48 h). The cells were processed for transmission electron microscopy to study the changes in steroidogenic functions of the cells. The effect of estradiol treatment was also evaluated on the quantity of androgen production and abundance of steroidogenic enzymes and proteins. The results indicated 17β-estradiol increased androgen production in ovarian stromal cells. In addition to enhanced androstenedione and testosterone production, estradiol stimulation was also based on the marked increase in abundance of mRNA transcript of steroidogenic enzymes [Star (Steroidogenic Acute Regulatory Protein), Cyp11a1, Cyp17a1, and hsd3b1 (3β-hydroxysteroid dehydrogenase)], as well as abundances of StAR and CYP11A1 protein. Thus, 17β-estradiol enhanced steroidogenesis in ovarian stromal cells. This study provided a basis for further exploration of regulation of steroidogenesis in ovarian stromal cells and the feedback mechanisms in association with estradiol.
Advances on hormone-like activity of Panax ginseng and ginsenosides
2020, Chinese Journal of Natural Medicines
Traditional Chinese medicine (TCM) has been paid much attentions due to the prevention and treatment of steroid hormone disorders. Ginseng, the root of Panax ginseng C. A. Meyer (Araliaceae), is one of the most valuable herbs in complementary and alternative medicines around the world. A series of dammarane triterpenoid saponins, also known as phytosteroids, were reported as the primary ingredients of Ginseng, and indicated broad spectral pharmacological actions, including anti-cancer, anti-inflammation and anti-fatigue. The skeletons of the dammarane triterpenoid aglycone are structurally similar to the steroid hormones. Both in vitro and in vivo studies showed that Ginseng and its active ingredients have beneficial hormone-like role in hormonal disorders. This review thus summarizes the structural similarities between hormones and dammarane ginsenosides and integrates the analogous effect of Ginseng and ginsenosides on prevention and treatment of hormonal disorders published in recent twenty years (1998–2018). The review may provide convenience for anticipate structure-function relationship between saponins structure and hormone-like effect.

View all citing articles on Scopus

View full text

A guide to understanding the steroid pathway: New insights and diagnostic implications

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Structure and naming of steroids

Steroid synthesis

Steroid synthesis and metabolism in the foetus and neonate

The alternative steroid pathway

Laboratory investigation of steroid biosynthesis

Normal physiological results

Conclusion

Funding

Financial disclosure

Conflict of interest

Contributor statement/disclosure summary

Acknowledgements

Adv Lipid Res

J Steroid Biochem Mol Biol

Adv Clin Chem

J Urol

J Biol Chem

Vitam Horm

J Steroid Biochem Mol Biol

Best Pract Res Clin Endocrinol Metab

Best Pract Res Clin Endocrinol Metab

Trends Endocrinol Metab

Am J Hum Genet

Clin Chim Acta

Clin Biochem

Steroids

Steroids

J Steroid Biochem Mol Biol

Clin Biochem

Clin Biochem

Mol Genet Metab

Steroids

Solid-phase radioimmunoassay of estradiol-17 beta

J Clin Endocrinol Metab

Microanalytical separation of steroids by gas chromatography

Nature

The Croonian lectures on the correlation of the functions of the body

Lancet

The Nobel Prize in physiology or medicine 1950: award ceremony speech

The development of cortisone as a therapeutic agent

Nobel lecture, December 11, 1950

Chemistry of the adrenal cortex hormones

Sterols. XI. 17α-hydroxy-11-desoxycorticosterone (Reichstein's substance S)

J Am Chem Soc

The nomenclature of steroids

Eur J Biochem

The nomenclature of steroids

Pure Appl Chem

The nomenclature of steroids

Cytochrome P450 reactions in the human brain

Atlas of steroid structure volume 2: published online in 2010

Biochemical education

Adrenal disease update 2011

J Clin Endocrinol Metab

Genital abnormalities mimicking congenital adrenal hyperplasia in premature infants

J Paediatr Child Health

11β-hydroxylase deficiency masked by alternative medicines

J Paediatr Child Health

Steroid profiling

Ann Clin Biochem

Mass spectrometry in the diagnosis of steroid-related disorders: clinical applications

Phase two steroid metabolism and its roles in breast and prostate cancer patients

Front Endocrinol

Steroid synthesis and catabolism in the fetus and neonate

Plasma cortisol, cortisone and urinary glucocorticoid metabolites in preterm infants

Biol Neonate

Maturation of the circadian rhythm of the adrenocortical functions in human neonates and infants

Horm Res

Activity of the adrenal fetal zone in preterm infants continues to term

Endocr Res

The human fetal adrenal: making adrenal androgens for placental estrogens

Semin Reprod Med

Transient anomalies in genital appearance in some extremely preterm female infants may be the result of foetal programming causing a surge in LH and the over activation of the pituitary-gonadal axis

Clin Endocrinol (Oxf)