133

THEMATIC REVIEW

The steroid metabolome of adrenarche

Juilee Rege and William E Rainey

Department of Physiology, Georgia Health Sciences University, 1120 15th Street, Augusta, Georgia 30912, USA

(Correspondence should be addressed to W E Rainey; Email: wrainey@georgiahealth.edu)

Abstract

Adrenarche is an endocrine developmental process whereby

humans and select nonhuman primates increase adrenal

output of a series of steroids, especially DHEA and

DHEAS. The timing of adrenarche varies among primates,

but in humans serum levels of DHEAS are seen to increase

at around 6 years of age. This phenomenon corresponds

with the development and expansion of the zona reticularis of

the adrenal gland. The physiological phenomena that trigger

the onset of adrenarche are still unknown; however,

This paper is one of three papers that form part of a thematic review

section on Adrenarche. The Guest Editor for this section was Ian Bird,

University of Wisconsin, USA.

Journal of Endocrinology (2012) 214, 133–1430022–0795/12/0214–133 q 2012 Society for Endocrinology Printed in Great

the biochemical pathways leading to this event have been

elucidated in detail. There are numerous reviews examining

the process of adrenarche, most of which have focused on

the changes within the adrenal as well as the phenotypic

results of adrenarche. This article reviews the recent and

past studies that show the breadth of changes in the

circulating steroid metabolome that occur during the process

of adrenarche.

Journal of Endocrinology (2012) 214, 133–143

Introduction

The human adrenal produces large quantities of the

19-carbon (C19) steroids DHEA and DHEAS during fetal

development, but the production of these steroids falls rapidly

after birth and remains low for the first few years of life.

Adrenarche refers to the re-emergence of adrenal C19 steroid

production at around 6 years of age that results from the

expansion and differentiation of the adrenal zona reticularis

(ZR; Cutler & Loriaux 1980, Miller 1988, 2009, Parker

1991, Auchus & Rainey 2004; Fig. 1). Neither DHEA nor

DHEAS are bioactive androgens, but they act as precursors for

production of more potent androgens including testosterone

in peripheral tissues that include hair follicles, genital skin, and

prostate (Kaufman et al. 1990, Rosenfield 2005, Pelletier

2008; Fig. 2). The initiation of androgen-dependent growth

of axillary and pubic hair (pubarche) is the phenotypic

hallmark of adrenarche (Ducharme et al. 1976, Auchus &

Rainey 2004, Havelock et al. 2004). The peripherally

converted bioactive androgens also stimulate the development

of apocrine glands in the skin to produce characteristic adult-

type body odor and can act on the sebaceous glands leading

to signs of acne (Rosenfield & Lucky 1993, Zouboulis et al.

2007). There are numerous reviews examining the process of

adrenarche, most of which have focused on the adrenarche-

associated changes in adrenal steroidogenic enzymes and

differentiation (Cutler & Loriaux 1980, Parker 1991, Auchus

& Rainey 2004, Havelock et al. 2004, Miller 2009, Nakamura

et al. 2009a). Herein, we have reviewed the recent and past

studies that show the breadth of changes in the circulating

steroid metabolome that occur during the process of

adrenarche.

C19 steroids and adrenal morphology

While most of our focus will be placed on the adrenarchal rise

in adrenal production of C19 steroids, it should be noted that

during development the fetal adrenal produces large amounts

of DHEAS. During this period, C19 steroids arise from a

developmental specific fetal zone that comprises 80% of the

fetal adrenal. Till birth, the fetal zone continues to secrete

dramatic quantities of the C19 steroid DHEAS, which is used

by the placenta for production of remarkably large amounts of

estrogens during pregnancy (Siiteri & MacDonald 1963,

Frandsen & Stakemann 1964, Bolte et al. 1966).

After birth, there is a dramatic involution of the fetal zone,

which accounts for the sharp decline in DHEAS synthesis in

the first few months of life (Fig. 1). The levels of DHEAS

remain low until adrenarche commences as a result of the

expansion of the ZR (Dhom 1973, Suzuki et al. 2000, Hui

et al. 2009). This phenomenon was first described and given

DOI: 10.1530/JOE-12-0183Britain Online version via http://www.endocrinology-journals.org

100

4020

6080

100120140160 500

450400350

50100150200250300

02 3 4 5

Age (years)

Serum DHEA and DHEASlevels across adrenarche

6 7 8 9 10 11 12 13 14 15

DH

EA

S (

µg

/dL

)

DH

EA

(n

g/d

L)

Figure 1 Serum DHEA and DHEAS levels before and after the onsetof human adrenarche. Based on steroid levels from de Peretti &Forest (1976, 1978).

J REGE and W E RAINEY . Steroid metabolome of adrenarche134

the name adrenarche by Albright et al. (1942) when they

observed the growth of axillary and pubic hair in the absence

of gonadal androgens due to congenital absence or

malformation of ovaries. Dhom (1973) characterized the

morphological changes that occurred in the prepubertal and

pubertal adrenal. According to this study, the adrenal cortex

of the infant shows distinct zona glomurerulosa (ZG) and

zona fasciculata (ZF) with little ZR. However, in the adrenals

of children around age 3 years, focal islands of ZR appear,

which expand at age 4–5 years (Dhom 1973, Hui et al. 2009).

A continuous layer of ‘functional’ ZR is first seen at age 6,

which continues to expand till age 12–13 years (Dhom 1973,

Hui, et al. 2009). The emergence of the developed ZR

corresponds with the rise in circulating DHEAS (Dhom 1973).

It has also been demonstrated that the thickness of the ZR is

directly proportional to the production of DHEAS (Dhom

1973, Reiter et al. 1977; Fig. 3).

Steroidogenic enzymes and cofactors involved inC19 steroid production

Although some steroidogenic enzymes and cofactor proteins

are common to all zones of the cortex, the zone-specific

production of steroids results in part due to differential

Figure 2 Adrenal-derived C19 steroids act as prandrogens in peripheral tissues including hair follpathway for bioactive androgen synthesis as wellhydroxyandrostenedione (11OHA) is shown. A,11OHT, 11b-hydroxytestosterone; 5,11OHT, 5a,117b-hydroxysteroid dehydrogenases (type 5 or typ

Journal of Endocrinology (2012) 214, 133–143

expression of key steroidogenic enzymes. The pathway

leading to the synthesis of DHEAS is quite simple and

requires only three steroidogenic enzymes. However, across

the period of adrenarche there are changes in the expression

pattern of the steroidogenic enzymes and cofactors that

facilitates C19 steroid production (Fig. 4A). Immunohisto-

chemistry studies have demonstrated the varied expression

of key steroidogenic enzymes within the cortical zones (Gell

et al. 1996, 1998, Suzuki et al. 2000, Hui et al. 2009).

The initial conversion of cholesterol to pregnenolone is an

essential step in all steroidogenic organs and is accomplished

by the enzyme cytochrome P450 cholesterol side-chain

cleavage (CYP11A1). This enzyme is expressed in all zones of

the adrenal and the expression pattern within the ZR does

not change during adrenarche (Nakamura et al. 2009a).

CYP17A1, a single steroidogenic enzyme localized in the

endoplasmic reticulum, is necessary for production of both

cortisol and DHEA as it catalyzes two biosynthetic activities:

17a-hydroxylase and 17,20-lyase (Yanase et al. 1991, Imai

et al. 1993, Brock & Waterman 1999). Thus, this enzyme is

needed for both ZF and ZR functions. However, Suzuki et al.

(2000) reported changes in expression of CYP17A1 in the

adrenal ZF and ZR of pre-adrenarche vs post-adrenarche

children. Their semiquantitative immunohistochemical

analysis showed that after age 5 years, the CYP17A1 protein

appeared to increase in both zones and reached a plateau level

after 13 years; suggesting that increased CYP17A1 could

impact the capacity for DHEA synthesis.

The expression of the flavoprotein mediating the dual

activity of CYP17A1, namely cytochrome P450 oxido-

reductase (CPR) has also been studied across adrenarche and

was found to increase in all the three adrenal zones, especially

the ZR (Suzuki, et al. 2000). The increased conversion of

17OHPreg to DHEA due to the elevated 17,20-lyase activity

of CYP17A1 (Katagiri et al. 1995) is central to understanding

the mechanisms underlying adrenarche. The lyase activity of

the enzyme is enhanced by the hemoprotein cytochrome b5

CYB5A; Katagiri et al. 1995, Auchus et al. 1998, Brock &

Waterman 1999, Dharia et al. 2004). CYB5A expression was

detected in the DHEAS-synthesizing fetal zone throughout

ecursors for the production of more potenticles, genital skin, and prostate. The classicalas a proposed alternative pathway using 11b-androstenedione; DHT, dihydrotestosterone;1b-hydroxytestosterone; 17bHSDs,e 3).

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Figure 3 DHEAS and expansion of the adrenal reticularis.(A) The relationship between the development of the ZRand plasma DHEAS levels (Dhom 1973, Reiter et al. 1977).(B) The morphological development of the adrenal zona reticularisdepicting the appearance of focal islands of reticular tissue and acontinuous ‘functional’ ZR (Dhom 1973).

Steroid metabolome of adrenarche . J REGE and W E RAINEY 135

gestation (Rainey et al. 2002, Dharia et al. 2004, Nguyen et al.

2008). Immunohistochemical studies indicated that CYB5A

is weakly synthesized in all the adrenal zones of pre-

adrenarchal children (Suzuki, et al. 2000). However,

CYB5A expression markedly rises in the ZR after age 5

years and plateaus at age 13 years, whereas the levels remain

extremely low in the ZG and ZF at all ages (Suzuki, et al.

2000). A reduction in the proportion of the adrenal cortex

that expresses CYB5A and DHEA levels was also detected

with aging (Parker 1999, Parker et al. 2000, Dharia et al.

2005). These finding shed light on the significance of CYB5A

in adrenarche.

The sulfoconjugation of steroids occurs extensively in the

adrenal cortex and is primarily carried out by the enzyme

SULT2A1, of the human hydroxysteroid sulfotransferase

family (Luu-The et al. 1996, Strott 2002, Shi et al. 2009).

SULT2A1 has a broad substrate specificity, which includes

DHEA, pregnenolone, and 17OHPreg. Sulfonation of

pregnenolone and 17OHPreg precludes metabolism by

HSD3B2 and CYP17A1 and thereby their utilization as

precursors for the mineralocorticoid, glucocorticoid, and

androgen biosynthetic pathways. Also, conversion of DHEA

into its sulfate ester, i.e. DHEAS, prevents the transformation

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of the poor androgen DHEA into more potent androgens.

Numerous studies have demonstrated that the transitional and

fetal zones of the human fetal adrenal abundantly express

SULT2A1 (Korte et al. 1982, Barker et al. 1994, Parker et al.

1994). Suzuki et al. (2000) carried out immunohistochemical

studies in the postnatal period from infancy to old age and

established that SULT2A1 became highly discernible in the

ZR at ages 5–13 years and reached a plateau thereafter; thus

confirming it as marker for ZR development.

The sulfonation of steroids by SULT2A1 requires a sulfate

donor, namely 3 0-phosphoadenosine 5 0-phosphosulfate

(PAPS; Weinshilboum et al. 1997, Strott 2002). In humans,

PAPS synthesis requires two isoforms of the enzyme PAPS

synthase, namely PAPSS1 and PAPSS2. Noordam et al. (2009)

examined an 8-year-old girl with early pubic and axillary

hair, and concluded that the cause of hyperandrogenism in

this subject was a set of compound heterozygous mutations

in PAPSS2. Mutated PAPSS2 generated a decreased amount

of PAPS, which is a prerequisite for steroid sulfonation. This

was consistent with impaired DHEA sulfonation; thereby

making the unconjugated DHEA pool available for conver-

sion into bioactive androgens such as testosterone.

During adrenarche, as the ZR expands, this zone was found

to have substantially lower levels of HSD3B2 compared with

the adjacent ZF (Fig. 4B). The relative lack of HSD3B2

expression/activity facilitates increased DHEAS synthesis

because HSD3B2 competes with CYP17A1 for pregnenolone

and 17OHPreg (Conley & Bird 1997, Rainey et al. 2002).

Thus, HSD3B2 expression in the expanding ZR coincides

with elevated adrenal DHEAS synthesis through the postnatal

period to adult life and is one of the driving forces of

adrenarche (Gell et al. 1996, 1998, Suzuki et al. 2000, Hui

et al. 2009). The phenomenon of increased androgen

production as a result of decreased HSD3B2 activity was also

demonstrated by McCartin et al. (2000) when they reported

the presence of premature adrenarche (PA) in an 11-year-old

subject with a profound loss of HSD3B2 activity owing to a

compound heterozygotic mutation in the HSD3B2 gene

(McCartin et al. 2000). However, it should be noted that there

are cortical cells located at the border between the ZF and

ZR in normal adult adrenal glands that co-express HSD3B2

and CYB5A, suggesting that these cells have the potential

for direct production of androstenedione (Nakamura et al.

2011). Human ovarian theca cells also co-express HSD3B2,

CYP17A1, and CYB5A and similarly have potential for

androstenedione biosynthesis (Dharia et al. 2004, Simard et al.

2005). Nevertheless, these results await further investigations

to clarify the regulatory mechanisms of co-expression of the

two enzymes in this population of cortical cells.

Control of adrenarche

The precise mechanisms that control adrenal androgen

biosynthesis have not been clearly defined. ACTH has been

considered as the accepted primary mediator of adrenarche

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Figure 4 (A) Steroid pathways for biosynthesis of adrenocortical steroids. StAR, steroidogenicacute regulatory protein; CYP11A1, cytochrome P450 cholesterol side-chain cleavage;CYP17A1, 17a-hydroxylase/17,20-lyase; HSD3B2, 3b-hydroxysteroid dehydrogenase type 2;CYB5A, cytochrome b5; HSD17B5, 17b-hydroxysteroid dehydrogenase type 5; SULT2A1,steroid sulfotransferase type 2A1; CYP11B1, 11b-hydroxylase. (B) Immunohistochemicalexamination of HSD3B2 expression in the human adrenal cortex from a 4-year-old child(pre-adrenarche) and a 9-year-old child (post-adrenarche). As observed in these micrographs,the post-adrenarche adrenal is characterized by the expansion of a HSD3B3-deficient ZR.

J REGE and W E RAINEY . Steroid metabolome of adrenarche136

for the past few decades (Rosenfeld et al. 1971a,b, Reiter et al.

1977). Clearly, dexamethasone suppression of adrenal

androgens suggests a regulatory role for ACTH (Abraham

1974, Kim et al. 1974, Rich et al. 1981). Moreover, children

with an ACTH receptor defect fail to experience adrenarche

(Weber et al. 1997), thereby supporting the postulate that

ACTH plays an essential role in this phenomenon. However,

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several studies have demonstrated that ACTH and cortisol

levels both remain constant even during adrenarche’s rise in

adrenal androgens (Mellon et al. 1991, Parker 1991, Penhoat

et al. 1991, Auchus & Rainey 2004). This is partly due to the

tight regulatory feedback system between ACTH and

cortisol, which keeps their levels within the physiologic

range throughout life (Reader et al. 1982, 1983) as opposed to

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Steroid metabolome of adrenarche . J REGE and W E RAINEY 137

DHEA and other C19 steroids that have no clear feedback

system. Thus, although ACTH affects both cortisol and

adrenal androgen secretion, there is a divergence in cortisol

and androgen production at adrenarche. A related hypothesis

proposing that the proximal 18-amino acid hinge region

(amino acids 79–96) of pro-opiomelanocortin (POMC) was a

stimulator for adrenal androgen synthesis, was not supported

by in vitro studies (Parker et al. 1989, Mellon et al. 1991,

Penhoat et al. 1991). However, there have been numerous

studies indicating that plasma levels of POMC-related peptides

like b-lipotropin and b-endorphin correlate to the increasing

levels of DHEAS during adrenarche (Genazzani et al. 1983a,b,

O’Connell et al. 1996). Several studies also indicated no

positive correlation between other potential endocrine

regulatory candidates including prolactin, insulin, and

insulin-like growth factor 1 with the DHEAS levels seen

during adrenarche (Aubert et al. 1974, Parker et al. 1978, Smith

et al. 1989, Guercio et al. 2002). Thus, determination of the

controlling factor of adrenarche still needs more investigation.

Anderson (1980) postulated that adrenarche is triggered

through an unknown mechanism involving the increased

intra-adrenal levels of cortisol associated with the growth of

the gland. Recent in vitro studies by Topor et al. (2011) also

established that cortisol inhibits HSD3B2 and stimulates the

biosynthesis of DHEA at concentrations above 50 mM.

Dickerman et al. (1984) and Byrne et al. (1985, 1986)

confirmed that there were age-dependent increases in intra-

adrenal concentrations of several C19 and C21 steroids and

proposed that these concentrations were in the range of

the Michaelis–Menten constant (Km) for HSD3B2 and

might therefore inhibit the enzyme and promote 17OHPreg

metabolism to DHEA at adrenarche. However, the same study

demonstrated that 1 mM cortisol did not inhibit HSD3B2

(Byrne et al. 1986). Also, DHEAS levels appear to be high in

adrenarchal children with untreated classic congenital

adrenal hyperplasia where intra-adrenal cortisol levels should

be low (Brunelli et al. 1995). Thus, the exact role for the

modulation of HSD3B2 enzymatic activity by intra-adrenal

steroid inhibitors remains unclear, while decreased expression

of HSD3B2 in the post-adrenarche ZR appears clear.

Steroid production in adrenarche

The difference in expression and activity of the various

adrenal steroidogenic enzymes from infancy to adulthood

suggest that there should also be age-related changes in serum

concentrations of several D5- and D4-steroids that would

underlie the physiologic processes of adrenarche.

Serum C21 steroids in adrenarche

As described earlier, it has been established that adrenarche is

associated with the expansion of the ZR having deficient

expression of HSD3B2 along with increasing expression of

CYP17A1. Also, 17OHProg and 17OHPreg are the key

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intermediates in cortisol and androgen metabolism, respect-

ively, thereby making them the most analyzed C21 steroids in

adrenarche. Abraham et al. (1973) demonstrated by RIA that

the concentration of 17OHPreg was higher in neonates than

in preadolescents, adult males, and nonpregnant women,

presumably due to the low activity of HSD3B2 in neonatal

adrenal cortex as compared with the adult cortex. Hughes &

Winter (1976) also observed age-related changes in serum

concentrations of 17OHProg. However, a detailed age-

dependent analysis of 17OHPreg and 17OHProg was

described by Shimozawa et al. (1988). They examined the

levels of 17OHPreg and 17OHProg by RIA in 11 umbilical

cord blood specimens and sera from 82 normal children of

various ages and 20 normal adults (Shimozawa et al. 1988).

According to this study, the levels of these steroids are the

highest in the cord blood owing to their metabolism by the

feto-placental unit; and after birth they start declining to reach

a nadir at 1–2 years age, followed by a gradual increase from

ages 3 to 6 years till adulthood. This is in agreement with the

observation that CYP17A1 expression in the ZF and ZR

increases during adrenarche (Suzuki et al. 2000).

The pattern of some of the unconjugated C21 steroids

has been traced in humans from birth to adulthood. Toscano

et al. (1989) examined the levels of serum pregnenolone

(50G7 ng/dl), cortisol (13G1.8 mg/dl), and 11-deoxy-

cortisol (52G3.6 ng/dl) in 20 children between ages 5.5and 9 years. Several studies carried out in large cohorts of

children between 2 and 12 years of age have established that

serum cortisol levels do not increase in adrenarche (Parker

et al. 1978, Franckson et al. 1980). Thus, it is clear that the

serum levels of most of the adrenal unconjugated C21 steroids

are not grossly affected during the course of adrenarche.

The robust expression of the adrenal-related sulfo-

transferase enzyme, namely SULT2A1 (Strott 2002), and its

broad substrate specificity has also encouraged researchers to

examine the sulfonated derivatives of various C21 steroids.

de Peretti & Mappus (1983) traced the levels of pregnenolone

sulfate (Preg-S) from birth to adulthood and observed that

Preg-S levels were high in cord plasma as a consequence

of residual fetal zone activity. The levels started declining

during the first year after birth and remained low thereafter.

Also, there was no detectable rise in Preg-S during the

adrenarchal period as is seen for the classical markers of

adrenarche, namely DHEA and DHEAS (de Peretti &

Mappus 1983). Shimozawa et al. (1988) demonstrated that

17OHPreg sulfate (17OHPreg-S) exhibited an age-related

change, wherein the 17OHPreg-S concentration was highest

in cord blood and decreased to a minimum at 3–6 years,

followed by a gradual increase from 7 years till adulthood

as opposed to its nonsulfated form that showed a nadir

at 1–2 years. These observations agree with what we now

know regarding the adrenarchal expansion of the ZR that

has high CYP17A1 and SULT2A1 and low HSD3B2

expression. This would explain the ability of 17OHPreg-S

to act as an additional steroid marker of adrenarche.

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J REGE and W E RAINEY . Steroid metabolome of adrenarche138

Serum C19 steroids in adrenarche

The weak C19 steroids DHEA and DHEAS are the

characteristic markers of adrenarche (Fig. 1). Extensive RIA

studies tracing these steroids from birth to adolescence

demonstrated that plasma DHEA levels rapidly declined in

the first 2 years of life, stayed low for the next few years, and

then dramatically increased after 6 years of age, corresponding

to ‘adrenarche’ (Ducharme et al. 1976, Korth-Schutz et al.

1976c, de Peretti & Forest 1976, 1978, Parker et al. 1978,

de Peretti & Mappus 1983). DHEAS has been the most

extensively studied steroid across adrenarche. A number of

research groups traced the age-related changes in plasma

DHEAS concentration and demonstrated that DHEAS

decreased slowly during the first years of life, remained low

till age 5 years and then abruptly started to rise (Korth-Schutz

et al. 1976a, Parker et al. 1978, de Peretti & Forest 1978, Rich

et al. 1981, Tung et al. 2004). Recent liquid chromatography–

tandem mass spectrometry (LC–MS/MS) studies in a large

cohort of children confirmed the same age-related pattern of

serum DHEA levels (Kushnir et al. 2010). The patterns seen

for both DHEA and DHEAS correlate well with intra-adrenal

changes occurring during this period. Specifically, the high

neonatal levels relate to the residual fetal zone that regresses

over the first year leading to a nadir of DHEA/DHEAS,

and their rise in the circulation correlates well with the

expansion of the reticularis.

The age-related patterns of secretion of several other C19

products have also been investigated but with varying results.

While some immunoassay studies suggest no adrenarchal rise

in androstenedione (Korth-Schutz et al. 1976c, Parker et al.

1978), others show significant adrenarche-related increases in

serum levels of androstenedione (Ducharme et al. 1976,

Franckson et al. 1980, Likitmaskul et al. 1995, Tung et al.

2004). Recent LC–MS/MS analysis also showed a similar

age-related rise in concentration of androstenedione (Kulle

et al. 2010). Parker et al. (1978) did not find a relationship

between serum 11b-hydroxyandrostenedione (11OHA)

concentration and age, whereas Franckson et al. (1980)

found that serum 11OHA increased with bone age

throughout prepubertal childhood. The major difference

between the studies was data analysis. Because of the circadian

association of 11OHA with cortisol, Franckson et al. utilized a

11OHA/cortisol ratio to make steroid comparisons.

Some immunoassay studies of testosterone in a large

population of children established that this androgen did not

rise demonstrably during the early stages of adrenarche

(Ducharme et al. 1976, Korth-Schutz et al. 1976c). Later

studies using LC–MS/MS confirmed that the plasma levels

of testosterone do not rise at adrenarche, but that its levels

do rise as puberty approaches in boys (Kulle et al. 2010). The

exact role of direct adrenal secretion of testosterone remains

unclear. Previous studies in adult women exhibiting signs of

androgen excess demonstrated that the adrenal could secrete

testosterone under pathologic conditions (Stahl et al. 1973a,b,

Greenblatt et al. 1976). In addition, the ZR expresses higher

Journal of Endocrinology (2012) 214, 133–143

levels of the enzyme 17b-hydroxysteroid dehydrogenase

type 5 (HSD17B5 or AKR1C3) than the adjacent ZF and

its expression increases across adrenarche (Hui et al. 2009,

Nakamura et al. 2009b). This enzyme has numerous

activities, including the ability to convert androstenedione

to testosterone (Fig. 4A). Thus, while it is clear that

DHEA/DHEAS represent the most consistent markers of

adrenarche, the production of more potent androgens needs

further study and the analysis of these androgens may require

consideration of adrenal circadian secretion as well.

Studies carried out in the 1970s and 80s (Mauvais-Jarvis

et al. 1973, Moghissi et al. 1984) indicating that the conjugated

androgen, androstane-3a,17b-diol glucuronide (Adiol-G)

would be a good measure of testosterone transformation,

led to further investigations that established the change in

plasma steroid glucuronide levels at different ages (Belanger

et al. 1986, Brochu & Belanger 1987). Brochu et al.

determined the levels of androsterone glucuronide and

Adiol-G across various age groups in males and concluded

that, since these 5a-reduced and conjugated steroids increase

significantly before puberty, the adrenal C19 steroids like

androstenedione and androstanedione must be getting

converted into androsterone glucuronide and potentially

into testosterone, which in turn is transformed into Adiol-G

during prepubertal development (Brochu & Belanger 1987).

Their findings support the premise that androgenic mani-

festations like appearance of pubic and axillary hair are

induced by the conversion of adrenal C19 steroids to active

androgens that are further metabolized into their conjugated

inactive products.

Urinary C19 steroids in adrenarche

Nathanson et al. (1941) was one of the first groups of

researchers to report the presence of androgen metabolites in

the urine of a large cohort of children. The results of their

study demonstrated that from ages 3 to 7 years, constant

amounts of androgens are excreted; however, at about 7 years,

the rate of excretion of these steroids begins to progressively

increase. Several studies were carried out to confirm the age-

related changes in androgen excretion. It was also demon-

strated that urinary total DHEA and DHEAS levels are

elevated from ages 8 to 12 years (Tanner & Gupta 1968,

Gupta 1970, Kelnar & Brook 1983). In 1991, the normal

ranges for urinary steroid excretion were determined for

the first time by gas chromatography (GC), and it was

demonstrated that urinary steroid excretion rates in childhood

positively correlate with growth and activity of the adrenal

cortex (Honour et al. 1991). With the advent of better

techniques and availability of commercial assays, Remer et al.

(1994a,b) demonstrated that urinary total 17-ketosteroid

sulfate and DHEAS concentrations in 8-year-old normal

children is significantly higher than preadrenarchal children

(aged 4 years). Extensive studies by GC–MS to determine the

urinary markers of adrenarche were carried out in a large

population of healthy children and adolescents between 3 and

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Steroid metabolome of adrenarche . J REGE and W E RAINEY 139

18 years (Remer et al. 2005), which established that DHEA

and its metabolites, like 16a-hydroxy DHEA, 3b,16a,17a-

androstenetriol, and androstenediol, show a continuous rise

from ages 3–4 years to 17–18 years, thereby suggesting

that adrenarche is a gradual process starting at an early age.

The gradual nature of increasing steroids would appear to

agree with Dhom’s histologic studies showing a gradual

expansion of the adrenal ZR (Fig. 3). Recent studies by Shi

et al. (2009) also confirmed the use of the C19 steroids as

urinary markers of adrenarche.

Future directions toward understanding theadrenarche steroid metabolome

Hui et al. (2009) suggested that the process of adrenarche

was associated with ZR increased expression of HSD17B5,

an enzyme able to convert androstenedione to testosterone.

They demonstrated the presence of HSD17B5 in the ZR of

human adrenals around age 9 years, which tracks well with

the onset of pubarche (Hui et al. 2009).

Testosterone derived from the testes as well as the adrenals is

converted to a more potent androgen, 5a-dihydrotetosterone

(DHT) by the enzyme 5a-reductases type I and II (SRD5A)

in androgen target tissues like genital skin and prostate

(Wilson et al. 1993, Chang et al. 2011). Recent studies

suggest that there are likely several pathways leading to

DHT biosynthesis (Wilson 1999, Wilson et al. 2003,

Auchus 2004, Ghayee & Auchus 2007, Fluck et al. 2011).

The peculiarity about these pathways is the synthesis

of 5a-androstane-3a,17b-diol (androstanediol) involving

5a-pregnane-3a,17a-diol-20-one as a key intermediate.

Androstanediol, in turn, acts as precursor for DHT, thereby

bypassing the need for testosterone, which is the ‘classical’

precursor for DHT production. Recently, Fluck et al.

(2011) confirmed that both the classical and alternative

pathways of testicular androgen production are involved

in the formation of normal human male sexual differen-

tiation, whereas another group suggested that synthesis

of DHT in castration-resistant prostate cancer requires

5a-androstenedione and not testosterone as an obligate

precursor (Chang et al. 2011).

Two derivatives of testosterone, 11b-hydroxytestosterone

(11OHT) and 11-ketotestosterone (11KT), may represent

unique adrenal-derived androgens. While testosterone and

DHT are the primary human androgens, 11OHT and 11KT

are the major androgens found in a variety of fish (Rosenblum

et al. 1985, Brantley et al. 1993, Yazawa et al. 2008). These C19

steroids are also produced in small quantities by mouse gonads

(Yazawa et al. 2008). The physiologic role of these steroids

in humans has not been studied, but circulating levels of

these androgens have been examined in human blood

(Kley et al. 1984, Schlaghecke et al. 1986). 11OHT could

be speculated to be an adrenal-derived androgen as it

might require the activity of 11b-hydroxylation via the

adrenal-specific enzyme CYP11B1 can be derived by the

www.endocrinology-journals.org

11beta-hydroxylation testosterone by the adrenal-specific

enzyme CYP11B1 in a reaction similar to that of the

conversion of androstenedione to 11OHA (Schloms et al.

2012, Kley et al. 1984, Schlaghecke et al. 1986). Because the

adrenal produces large amounts of DHEA, DHEAS,

androstenedione, and 11OHA, it is likely that

these androgen precursors will feed into both the classical

and alternative peripheral metabolic pathways that can lead

to active androgens.

Premature adrenarche

PA can be defined as the early rise in adrenal androgen

production that usually results in the appearance of pubic or

axillary hair before age 8 years in girls and 9 years in boys,

without the appearance of other secondary sex characteristics

(Talbot et al. 1943, Silverman et al. 1952). Girls with PA

are susceptible to the development of polycystic ovary

syndrome with hirsutism, irregular menses, and hyperan-

drogenism (Ibanez et al. 1993). PA is also an early onset

predictor of hyperinsulinemia (Oppenheimer et al. 1995,

Ibanez et al. 2000). Children with PA have been shown to

exhibit higher serum levels of DHEA, DHEAS, androstene-

dione, and testosterone as well as their urinary metabolites

(Doberne et al. 1975, Korth-Schutz et al. 1976b,c, Rosenfield

et al. 1982, Voutilainen et al. 1983, Rosenfield & Lucky 1993,

Likitmaskul et al. 1995, Ibanez et al. 2000). Recently studied

LC–MS/MS steroid profiles of infants with fine genital hair

showed a mild elevation of DHEAS as compared with healthy

pre-adrenarchal children, thus indicating that pubic hair in

infancy may represent a mild and early-onset variant of

PA (Kaplowitz & Soldin 2007). Toscano et al. observed

increases in plasma levels of C21 steroids like pregnenolone

and 17OHPreg along with the C19 steroids like DHEA,

DHEAS, and androstenedione but with no change in cortisol

or 11-deoxycortisol in PA children as compared with

controls. They interpreted these steroid changes to suggest

that the PA adrenal had decreased HSD3B2 activity and

increased CYP17A1 activity (Toscano et al. 1989). They also

indicated that PA might not be exclusively dependent on

ACTH regulation (Toscano et al. 1989). The levels of

Adiol-G also increased in children with PA and showed a

strong correlation with DHEA, DHEAS, and androstene-

dione (Montalto et al. 1990, Riddick et al. 1991, Balducci

et al. 1992, 1993, Ibanez et al. 2000). Thus, it is clear that

numerous adrenal-derived steroids can be used as markers in

the diagnosis of PA.

Conclusions

Adrenarche is characterized by rising levels of C19 steroids

like DHEA and DHEAS due to the expanding adrenal ZR,

and in humans this occurs at ages 6–8 years. The phenotypic

hallmark of adrenarche is the appearance of axillary and pubic

Journal of Endocrinology (2012) 214, 133–143

J REGE and W E RAINEY . Steroid metabolome of adrenarche140

hair. This phenomenon is driven by the concerted actions

of the enzymes CYP17A1, SULT2A1, and the cofactor

CYB5A, which are highly expressed in the expanding

population of ZR cells seen during adrenarche. The low

expression of HSD3B2 in the ZR also enables the

increased production of androgens by the adrenal cortex.

Herein, we have reviewed recent and past studies that discuss

the changes in the circulating steroid metabolome that occur

during the process of adrenarche. It is clear that along with

DHEA and DHEAS, serum levels of other C19 steroids like

androstenedione, 11OHA, and Adiol-G are elevated during

adrenarche. The serum levels of most of the adrenal

unconjugated C21 steroids also do not seem to be excessively

affected during the course of adrenarche. It is still worth

noting that although biochemical pathways leading to the

formation of these steroids have been elucidated in detail, the

primary signal(s) that drive adrenarche remains unknown.

This makes the process of adrenarche one of the least

understood of human endocrine developmental events.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived

as prejudicing the impartiality of the research reported.

Funding

This work was supported by grants from the National Institute of Health

(DK069950) to W E R.

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Received in final form 14 June 2012Accepted 19 June 2012Made available online as an Accepted Preprint19 June 2012

Journal of Endocrinology (2012) 214, 133–143