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 Preface
The human body is primarily governed by two intri-
cate communicating networks: the nervous system
and the endocrine system. The study of the interrela-
tionship of these two networks created the discipline
of neuroendocrinology. Recent advances in this field
have transformed our view of how human endocrine
homeostasis is maintained. For example, the discov-
ery of the adipokine leptin revolutionized our under-
standing of the neural mechanisms by which we
regulate body weight. A further case is the discovery
of the KISS1 gene, and its encoded neuropeptide kis-
speptin, now recognized as obligatory for successful
human reproductive function.
Although several texts are currently available that
cover the field of clinical neuroendocrinology, they
are almost exclusively advanced, multi-author books
written by experts and largely aimed at medical spe-
cialists. While these texts provide a comprehensive
clinical and basic science review of the subject, there
is a compelling need for an introductory description
of the human neuroendocrine system in health and
disease. Our book is therefore designed to emphasize
the key physiological principles necessary for an
understanding of various clinical neuroendocrine
disorders.
Introductory chapters discuss the fundamentals
that govern how the hypothalamic–pituitary system
interacts with various endocrine target tissues.
Topics include cellular communication, hormone
receptor systems, hormone assays and a description
of the importance of hormonal secretory rhythms.
Subsequent chapters outline the essentials of human
female reproduction, the regulation of body weight
and metabolism with a focus on obesity, the control
of prolactin secretion and the principles of adrenal,
thyroid and growth hormone physiology. Finally, in
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a separate chapter on sellar masses, key elements of
clinical history, biochemical and radiological assess-
ment as well as epidemiology of sellar masses are
discussed. The clinical implications of various phy-
siological principles as well as cases from our clinics
are included in the text. The text itself is liberally
illustrated with full-color, high-resolution images to
provide concise summaries of information. Extensive
lists of references emphasize original papers based
on human data.
We intend the book to be useful at multiple
levels, though it is especially aimed at those stu-
dents and clinicians not previously exposed to
a specific course in neuroendocrinology. For exam-
ple, senior medical students making decisions to
pursue a specialty will find it helpful, as will those
residents and clinical fellows who are embarking on
their chosen fields. In addition, this book should
provide a crucial clinical context for biomedical
science graduate students who may already be
familiar with basic science research principles.
We include an extensive and up-to-date reference
list for each chapter, and additional material is
provided under a further reading list. The latter
tend to be clinical reviews that may be particularly
useful to the more advanced reader and will provide
a convenient link to the available specialized texts.
A selection of review questions is provided at the
end of each chapter.
The field of neuroendocrinology is a rapidly evol-
ving area of health sciences. This introduction to
clinical neuroendocrinology should serve as a guide
to medical students, clinicians and biomedical
science students, as well as their teachers, in nego-
tiating a fascinating and essential clinical field of
study.
ix
 Chapter
Basic Principles in Clinical

1 Neuroendocrinology I: Receptor
Mechanisms

The homeostatic functions of the body are primarily
controlled by neuronal cells communicating through
electrical impulses and endocrine cells communicat-
ing through chemicals. Neuroendocrinology is the
branch of endocrinology that is concerned with how
the brain regulates the endocrine milieu.
An essential and critical characteristic of this neural
control is that endocrine hormones have profound
effects on brain function through homeostatic feed-
back systems. An understanding of the chemical
mechanisms that underpin neuroendocrine regula-
tion is critical when dealing with clinical disorders
of the neuroendocrine system. For example, the
clinical complications of abnormal growth, thyroid
disorders, obesity and Cushing’s syndrome can be
confronted through addressing the underlying neu-
roendocrine principles.
For the most part, the brain influences endocrine
targets in tandem with the pituitary gland; that is,
pituitary hormone secretion is directed by various
stimuli secreted from hypothalamic neurons. Thus,
luteinizing hormone (LH) is released from pituitary
gonadotropes following stimulation by gonadotropin
releasing hormone (GnRH), a neuropeptide produced
by hypothalamic neurons. Figure 1.1 illustrates the
hypothalamic releasing hormones that regulate ante-
rior pituitary hormone secretion. Note that dopa-
mine – a neurotransmitter – controls prolactin
(PRL) secretion. In contrast, posterior pituitary hor-
mones, such as oxytocin, are not under the influence

HYPOTHALAMUS

Thyrotropin releasing hormone

Growth hormone releasing hormone
Dopamine
Gonadotropin releasing hormone
Somatostatin ANTERIOR
Corticotropin releasing hormone PITUITARY

Growth hormone
Luteinizing hormone
Follicle stimulating hormone
Adrenocorticotropic hormone
Thyroid stimulating hormone
POSTERIOR Prolactin
PITUITARY

Vasopressin
Oxytocin

Figure 1.1 Hypothalamic and pituitary hormones of the neuroendocrine system. Public domain images from Ladyofhats: http://en.wikipedia
.org/wiki/File:Endocrine_central_nervous_en.svg.

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 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms
Regulatory
neurons
+
–
– tyrosine kinase-dependent receptors. An additional,
± –
GnRH
neurons
Infundibular
nucleus
GnRH
Portal
vasculature
± –
Anterior
pituitary
LH/FSH
Estradiol Testosterone
Progesterone
Ovary Testis
Figure 1.2 Homeostatic control of GnRH secretion and the reproductive
system. Schematic diagram highlighting the role of GnRH neurons in the
control of human reproduction. Red arrows indicate the positive and
negative feedback effects of serum estradiol and progesterone exerted
on GnRH secretion. This control is imposed at several levels: directly on
GnRH neurons; at the level of gonadotrophs; and on neurons (stimula-
tory and inhibitory) that regulate GnRH neurons. Blue arrows illustrate
the negative feedback effects of testosterone on GnRH release in the
male. Abbreviations: GnRH, gonadotropin releasing hormone; LH,
Luteinizing hormone; FSH, Follicle stimulating hormone.
of releasing hormones but instead are secreted
directly from hypothalamic neuron terminals in the
posterior pituitary.
A typical neuroendocrine feedback network is
exemplified by the reproductive system in which the
target organs (ovary and testis) respond to gonado-
tropin stimulation by releasing sex hormones (estra-
diol, progesterone or testosterone) that, in turn, exert
negative feedback on the hypothalamus to influence
secretion of GnRH and LH/follicle stimulating hor-
mone (FSH) (see Figure 1.2 and Chapter 3). Other
examples will be described in subsequent chapters.
The target organ sensitivity to stimulation, and the
neuronal response to hormonal feedback, is depen-
dent upon a variety of receptor mechanisms. For
example, in Figure 1.2, the response of pituitary gona-
dotrophs to stimulation with the peptide GnRH is
2
governed by specific membrane receptors of the
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G protein-coupled receptor (GPCR) superfamily.
Other hormones, such as leptin, bind to so-called
intracellular, superfamily of steroid hormone recep-
tors mediates the feedback effects of steroid hormones
such as estradiol and cortisol at the level of the ante-
rior pituitary and hypothalamus. Thus, an interplay of
fluctuating hormonal levels with receptor sensitivity
dictates homeostatic neuroendocrine regulation.
The following sections will summarize the receptor-
signaling systems that: (a) regulate target organ activ-
ity, for example, stimulation of gonadotrophs by
GnRH acting through cell surface GPCRs; and (b)
control hormonal feedback in the hypothalamus and
pituitary, for example, intracellular estradiol recep-
tors in brain and pituitary.
1.1 Cell Membrane Receptors
Peptide hormones, neuropeptides and neurotrans-
mitters are generally water soluble and cannot easily
enter their target cells. They regulate cellular activity
by binding to specific receptors located in the plasma
membranes of their target cells. In order to induce
biochemical changes within the target cell, they act as
first messengers to activate an intracellular second
messenger, such as cyclic adenosine monophosphate
(cAMP). The transduction of information from the
first to the second messenger is accomplished through
the activation of membrane proteins (e.g., G proteins)
and enzymes, such as adenylate cyclase.
This section illustrates the role of membrane recep-
tors for peptide hormone and neurotransmitter action,
the mechanisms by which signal transduction across
the cell membrane occurs, the role of G proteins and
receptor tyrosine kinases in this signal transduction,
and the second messenger systems activated.
1.2 G Protein-Coupled Receptors
GPCRs are characterized by their seven transmem-
brane domain structures attached to trimeric
G proteins (Figure 1.3). They bind multiple neuro-
transmitters, hormones and peptides, and control
almost all known physiological processes, including
neuroendocrine regulation, cardiovascular function,
behavior and immune function. A variety of
G proteins together with receptors, effectors and var-
ious regulatory intracellular proteins are the compo-
nents of a complex and versatile signal transduction
system (for a detailed review see Wettschureck and
 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

GnRH
CRH GHRH
TSH
GPCR
dopamine

Activated G protein

LIN
Extracellular

RE
5 6 7

SO
GH
4

M
Cytoplasm
12 3 +
γ γ
Gα Gα
GDP β
GTP
β +
Gαs Gαq –

Gαs cAMP
AC PLC cAMP/Ca2+ Inhibition of
PLC/IP3/Ca2+ cAMP
Gαq PLC
cAMP
Gαi cAMP

PKA PKC
+ –

GH gene Inhibition
Biological Gene expression
expression; of
response
GH release GH secretion

Nucleus DNA

Figure 1.3 Schematic diagram of GPCR signaling via G proteins

and second messengers. GPCRs, in the inactive state, possess seven
transmembrane domains coupled to a G protein complex consisting
of α-, β- and γ-subunits, plus a molecule of GDP. Binding of a specific
ligand activates the Gα-subunit by replacing GDP with GTP, followed
by dissociation of the βγ-subunit. This step is reversible following
dissociation or degradation of the receptor stimulus. The Gα-subunit
exists as three forms: (a) a stimulatory Gαs that increases cAMP
production and the activation of protein kinase A; (b) a stimulatory
Gαq that selectively stimulates the PLC pathway to activate PKC; and
(c) an inhibitory G03B1i pathway that inhibits cAMP production.
The protein kinases may regulate enzyme activity or gene expression
via transcription factors (dotted arrows) that bind to target genes.
Examples of ligands that bind to GPCRs are GnRH, CRH, TSH and
dopamine. Image reproduced with permission (Neumann et al., 2014).
Abbreviations: AC, adenylate cyclase; GDP, guanosine diphosphate;
GnRH, gonadotropin releasing hormone; GPCR, G protein-coupled
receptor; GTP, guanosine triphosphate; PIP2, phosphatidylinositol 4,5
bisphosphate; PKC, protein kinase C; PLC, phospholipase C.
GH SECRETION
SOMATOTROPH
Figure 1.4 GPCR-dependent mechanisms regulating GH secretion
from pituitary somatotrophs. Receptors for GHRH and ghrelin are
stimulatory G protein coupled, linked to cAMP/PKA and PLC/PKC
signals, respectively. GHRH and ghrelin act synergistically to induce
intracellular calcium ion mobilization that, in turn, controls GH
secretion. These stimuli also induce GH gene expression and GH
synthesis in somatotrophs. In contrast, SOM binds to an inhibitory
GPCR that reduces the accumulation of cAMP, decreasing the release
of GH(Ben-Shlomo and Melmed, 2010). Abbreviations: GPCR,
G protein-coupled receptor; IP3, inositol triphosphate; PKA, protein
kinase A; PKC, protein kinase C; SOM, somatostatin.

Offermanns, 2005). For the purposes of this chapter,
Figure 1.3 outlines, in general terms, the processes by
which extracellular stimuli are rapidly transduced to
intracellular signals that ultimately control gene
expression and biological response. The figure
includes typical neuroendocrine stimuli (GnRH, cor-
ticotropin releasing hormone [CRH], thyroid stimu-
lating hormone [TSH] and dopamine) that act via
GPCRs. Other examples will be covered in subsequent
chapters.
By way of illustration, the pituitary somatotroph is
a cell type that utilizes all three of the GPCRs illu-
strated in Figure 1.3; that is, ghrelin and growth hor-
mone releasing hormone (GHRH) synergize to
stimulate growth hormone (GH) secretion, whereas
somatostatin serves to inhibit GH secretion
(Figure 1.4). This system is covered in more detail in
Chapter 8. A further example is the synergistic stimu-
lation of adrenocorticotropin (ACTH) from pituitary
corticotrophs by CRH and vasopressin (see Chapter 5;
Figure 5.5)
Clinical Significance of GPCRs
G protein receptors are firmly implicated in all the
hormonal systems described in this book. For exam-
ple, Figure 1.3 illustrates that important neuroendo-
crine molecules such as GnRH (reproductive system),
CRH (regulation of the hypothalamic–pituitary–adre-
nal system), TRH (hypothalamic–pituitary–thyroid
regulation) and dopamine (PRL secretion) all func-
tion through specific GPCRs. Other examples will be
covered in later chapters. These receptors represent
3

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 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

a superfamily of human membrane proteins. Drugs Desensitization is reversible by replacing continuous

that target them account for approximately 30% of the
global market share of therapeutic drugs, with esti-
mated sales for 2011–2015 of approximately
US$890 billion (Hauser et al., 2017).
The responsiveness of GPCRs to stimulation may
be compromised in some neuroendocrine disorders.
Receptor function is affected in two principal ways:
(a) when GPCRs are subjected to chronic stimulation
to produce tachyphylaxis, and (b) when mutations
occur in the genes for receptor proteins or
G proteins.
Chronic Stimulation and GPCR Downregulation
For example, continuous stimulation of the human
female pituitary with the releasing hormone GnRH
downregulates (desensitizes) the normal response
such that LH secretion and ovulation is inhibited
(Southworth et al., 1991). A major consequence of
continuous stimulation of gonadotrophs is inhibition
of the normal response through the loss of cell surface
GnRH receptors (Engel and Schally, 2007).
GnRH treatment with an episodic, pulsatile stimula-
tion. This will be described in more detail in
Chapter 3. A similar phenomenon is reported with
stimulation of LH secretion with the neuropeptide
kisspeptin, which also binds to a GPCR (Jayasena
et al., 2009; see Chapter 3).
Mutations in GPCR Genes
The clinical significance of GPCRs in the neuroendo-
crine system is reinforced when taking into account
endocrine/neuroendocrine disorders that result from
loss-of-function or gain-of-function mutations in the
genes for receptor proteins or G proteins (Vassart and
Costagliola, 2011). Table 1.1 lists a group of mutations
of GPCRs relevant to endocrine diseases, together
with the appropriate chapter where these receptors
are discussed (based on data from Lania et al., 2006;
Vassart and Costagliola, 2011). A detailed discussion
of gain-of-function GPCR mutations associated with
endocrine disorders is covered elsewhere (Fukami
et al., 2018).

Table 1.1 Clinical and biochemical features of endocrine disorders caused by GPCR mutations

Clinical features Biochemical feature GPCR affected (type of

mutation)
Thyroid disorders (Chapter 6)
Isolated central hypothyroidism with normal Normal or low TSH, low fT4 absent TRH receptor (inactivating)
pituitary MRI TSH and prolactin responses to TRH
test
Complete congenital hypothyroidism High TSH, low fT4, no goiter, no TRH receptor (inactivating)
antibodies to thyroglobulin or
thyroperoxidase
Juvenile hyperthyroidism with goiter Low TSH, high fT4, no antibodies to TSH receptor (activating)
thyroglobulin or TSH receptor
Reproductive disorders (Chapter 3)
Delayed puberty Low sex steroids and low–normal Kisspeptin receptor (inactivating)
LH and FSH responsive to GnRH test
Low sex steroids and low–normal GnRH receptor (inactivating)
LH and FSH poorly responsive to
GnRH test
Primary amenorrhea with normal development of High LH, normal or high FSH, and LH receptor (inactivating)
primary and secondary sexual characteristics low estradiol and progesterone
Male precocious puberty with normal pituitary High testosterone and low LH and LH receptor (activating)
MRI FSH with prepubertal response to
GnRH test
Primary or early-onset secondary amenorrhea, High LH, high FSH FSH receptor (inactivating)
variable development of secondary sex
characteristics and premature arrest of follicular
maturation
Ovarian hyperstimulation syndrome during in vitro None FSH receptor (activating)
4 fertilization

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 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Table 1.1 (cont.)

Clinical features Biochemical feature GPCR affected (type of

mutation)
Obesity (Chapter 4)
Early-onset or severe adult obesity, associated with Hyperinsulinemia Melanocortin 4 receptor
hyperphagia (inactivating)
Growth disorders (Chapter 8)
Dwarfism associated with abdominal adiposity Low GH and insulin-like growth GHRH receptor (inactivating)
factor 1, unresponsive to GHRH test
Water balance disorders (Chapter 9)
Nephrogenic diabetes insipidus Hypernatremia, low urine V2R (inactivating)
osmolality, normal or high VP
Nephrogenic syndrome of inappropriate Hyponatremia, low serum V2R (activating)
antidiuresis osmolality, inappropriately high
urine osmolality, undetectable VP
levels

Abbreviations: FSH, follicle stimulating hormone; fT4, free thyroxine; GH, growth hormone; GHRH, growth hormone releasing
hormone; GnRH, gonadotropin releasing hormone; GPCR, G protein-coupled receptor; LH, luteinizing hormone; MRI, magnetic
resonance imaging; TRH, thyrotropin releasing hormone; TSH, thyroid stimulating hormone; VP, vasopressin; V2R, vasopressin
receptor 2.

1.3 Tyrosine Kinase-Dependent LEPTIN

Leptin receptor dimer

Receptors Plasma membrane

Peptide hormones such as leptin, PRL and GH (see
JAK JAK
Chapters 4, 7 and 8, respectively) do not bind to Cytoplasm P P
GPCRs. As shown in Figure 1.5, the receptor struc- P P

tures – in this case the leptin receptor dimer – have P P SOCS3

only two transmembrane domains. Leptin binding to STAT STAT

–

a single transmembrane protein induces receptor

dimerization, which then stimulates a tyrosine kinase
P STAT
pathway rather than the G protein signaling shown in
P
Figure 1.3. For leptin, PRL and GH, this is the second STAT

messenger JAK/STAT pathway (for details of the GH

system see Chapter 8; Figure 8.12). Nucleus
STAT
For example, binding of leptin to its receptor acti- P
Gene
P
vates (phosphorylates) Janus kinase (JAK) proteins STAT expression

that are docked on the intracellular domain.

Activated JAKs then phosphorylate the signal trans-
ducer and activator of transcription (STAT) family of
transcription factors. Dimerization of STAT proteins
precedes translocation to the nucleus where binding
to response elements on DNA modulates transcrip-
tion of target genes. Unlike the leptin receptor, the
PRL and GH receptors exist as preformed dimers (see
Figure 8.12 for GH, and Brooks and Waters, 2010) but
also employ the JAK/STAT pathway following bind-
ing of the ligand (Bernard et al., 2015).
Figure 1.5 Leptin receptor and the JAK/STAT pathway. Binding of
leptin to its receptor dimer activates (phosphorylates; P) JAK
proteins. Activated JAKs then phosphorylate the STAT family of
transcription factors. Dimerization of STAT proteins precedes
translocation to the nucleus where binding of the transcription
factor to response elements on DNA modulates transcription of
target genes. Also included is a negative feedback system where
gene expression of SOCS3 modulates leptin stimulation. Image
reproduced with permission (Dodington et al., 2018). Abbreviations:
STAT, signal transducer and activator of transcription; SOCS3,
suppressor of cytokine signaling 3.
5

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 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Clinical Significance of Tyrosine insensitivity – mutations in the intracellular STAT5

Kinase-Dependent Receptors
The clinical importance of leptin, PRL and GH is
described in the appropriate chapters. The clinical con-
sequences of abnormal receptor signaling are pro-
found, and defects, either at the receptor or in the
intracellular pathway, appear as a hormone deficiency.
For example, as described in Chapter 4, obese indivi-
duals are insensitive to their own high levels of leptin.
By analogy with the insulin resistance seen in type 2
diabetes, the common forms of diet-related obesity are
thought to be attributable to “leptin resistance,” a state
in which multiple cellular processes block leptin recep-
tor signaling (Myers et al., 2010). Thus, high levels of
endogenous leptin, derived from the increased fat
mass, fail to reduce food intake or body weight. Also,
patients with mutations in the leptin receptor are
severely obese and effectively leptin-free, even though
leptin levels are high (Farooqi et al., 2007). In these
cases, leptin is unable to signal to downstream path-
ways. Due to loss-of-function mutations in the GH
receptor (Rosenfeld et al., 2007; Brooks and Waters,
2010), patients insensitive to GH (Laron syndrome)
show severe growth failure and insulin-like growth
factor 1 (IGF-1) deficiency (see Chapter 8). Over sixty
loss-of-function mutations in the GH receptor have
been reported, although a new cause of GH
gene (see Figure 8.12) – is now described; that is, the
intracellular signaling pathway is defective (Hwa,
2016). Loss-of-function mutations in the PRL receptor
have also been described (Bernard et al., 2015). Such
patients present with hyperprolactinemia, possibly due
to the inability of PRL to exert negative feedback on
pituitary lactotrophs.
1.4 Intracellular Receptors for
Steroid Hormones
Steroid hormones (e.g., testosterone, estradiol and
cortisol; Figure 1.6) and thyroid hormones (see
Figure 6.5) are fat-soluble molecules transported in
the blood bound to carrier proteins.
Steroid hormones readily diffuse through cell
membranes into any cell in the body, but only their
target cells, in brain and pituitary for example, possess
specific intracellular receptors. These receptors have
common structural elements and belong to
a superfamily of receptor proteins (Figure 1.7).
Each receptor protein contains a hormone binding
domain (HBD) that is specific for each hormone.
Thus, for example, the human glucocorticoid receptor
(hGR) binds cortisol in the HBD, whereas the proges-
terone receptor (hPR) recognizes only progesterone.
Binding of the hormone to its specific receptor

Figure 1.6 Chemical

structures of the major steroid
hormones. Four principal
families of steroid hormones
are: androgens (e.g.,
testosterone), estrogens (e.g.,
estradiol), glucocorticoids
(e.g., cortisol) and mineralo-
corticoids (e.g., aldosterone).
They are all derived from
cholesterol. An important
family not shown is the
progestogens (e.g.,
progesterone). Note that the
female sex hormone estradiol
is formed by aromatization
from the male hormone
testosterone. Copyright
A. Pincock.

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 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

hAR 100
NTD
100
DBD H HORMONE BINDING DOMAIN
100

hERα NTD DBD H HORMONE BINDING DOMAIN

15 51 20

hERβ NTD DBD H HORMONE BINDING DOMAIN

15 56 22

hGR 15
NTD
77
DBD H
50
HORMONE BINDING DOMAIN

hPR 15
NTD
80
DBD H
53
HORMONE BINDING DOMAIN

Figure 1.7 Structures of members of the human steroid receptor superfamily. These intracellular receptors consist of a single polypeptide chain
and all of the family members contain a hormone binding domain that is specific for each hormone. For example, hGR binds cortisol in
this region, whereas the hPR will only recognize progesterone. The DBD enables the hormone-receptor complex to bind to the HRE (see
Figure 1.8) on a target gene. The hinge region (H) is important, along with the DBD, for nuclear localization of the receptor, and the NTD is
crucial for the activation of gene transcription once the hormone-receptor complex reaches the nucleus and binds with the HRE. Numbers
indicate the degree of structural homology in each domain, compared with the hAR (McEwan and Brinkmann, 2016). Abbreviations: DBD, DNA
binding domain; hAR, androgen receptor; hERα, estrogen receptor α; hERβ, estrogen receptor β; hGR, glucocorticoid receptor; hPR,
progesterone receptor; HRE, hormone response element; NTD, amino terminal domain.

enables the DNA binding domain (DBD) to interact Estrogen Receptors

with specific sites on target genes called hormone
It is beyond the scope of this text to provide a description
response elements (HRE; see Figure 1.8). The hinge
of the clinical role of all of the nuclear receptors illu-
region (H) appears to be important, along with the
strated in Figure 1.7, and the estrogen receptor (ER) will
DBD, for nuclear localization of the receptor, and the
be used here as an example. ERs exist in three forms, two
amino terminal domain (NTD) is crucial for the acti-
of which are nuclear (ERα and ERβ, also termed ESR1
vation of gene transcription once the hormone-
and ESR2; Figure 1.7) and the third, located in cell
receptor complex reaches the nucleus and binds with
membranes, is a GPCR. The latter – termed the
the HRE (McEwen and Brinkmann, 2016). Note that
G protein-coupled ER-1 (GPER-1) – is a relative new-
the receptor proteins have no biological activity until
comer to ER physiology, and these receptors permit
they bind to a hormone; that is, the hormone-receptor
rapid, non-genomic (non-nuclear), cellular responses
complex acts as a transcription factor at specific sites
to estrogen treatment. There is evidence of
(HRE) on target genes. Figure 1.8 illustrates the
a physiological role for GPER-1 in the reproductive,
sequence of events that occur when a steroid hormone
nervous, endocrine, immune and cardiovascular sys-
diffuses into target cells, such as pituitary or hypotha-
tems (Prossnitz and Barton, 2011), and GPER-1 appears
lamus. The hormone is released from the binding
to be a promising and novel therapeutic target and
globulin (BG), enters the cell and binds to a specific
prognostic indicator (Barton, 2016). Membrane recep-
receptor (R) in the cell cytosol. The unoccupied R is
tors for androgens, glucocorticoids (GCs), aldosterone
coupled to a so-called molecular chaperone (heat
and thyroid hormone have also been described.
shock protein 90; HSP90) that ensures the receptor
ERα and ERβ, the products of two distinct genes,
is stabilized in the correct shape. Following hormone
are localized in many tissues (Figure 1.9), with ERα
binding to the receptor-HSP90 complex, the HSP90
especially concentrated within the reproductive sys-
dissociates and the remaining hormone-receptor
tem (hypothalamus, pituitary, breast and uterus).
complex dimerizes. The dimer then enters the cell
Details of their functional role in estrogen feedback
nucleus where it attaches to an HRE to modify gene
and the regulation of anterior pituitary secretion of
expression and the export of mRNA into the cell
gonadotropins through the menstrual cycle is out- 7
cytosol where it is translated into protein.
lined in Chapter 3.
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 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms
This figure reveals that ERs are implicated in reg-
ulating multiple complex physiological processes in
humans, and abnormal ER function leads to a variety
of diseases, such as cancer, metabolic and cardiovas-
cular disease, neurodegeneration, inflammation and
osteoporosis (Jia et al., 2015).
Clinical significance: Using the menopause as
a specific example, estrogen deprivation has profound
effects particularly in the central nervous system; for
example, vasomotor symptoms such as hot flashes
and night sweats, development of anxiety, depression,
poor quality of sleep and migraine. Figure 1.10 illus-
trates the widespread influence of estrogen depletion
(Monteleone et al., 2018).
Estrogen replacement at the menopause inevitably
influences many systems, some not without risk, as in
the possibility of breast cancer (Warner et al., 2017).
Selective targeting of ERα and ERβ appears to be
a promising way to achieve beneficial estrogenic
effects while avoiding unwanted side effects. ERβ
selective agonists are now available that have no effect
in breast tissue but may be therapeutic agents consid-
ered for prevention and treatment of cancer, meta-
8 bolic and cardiovascular diseases, neurodegeneration,
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Figure 1.8 Schematic view of a steroid
hormone interacting with a target cell.
The hormone is reversibly bound to
a binding globulin (BG) before the free
hormone freely diffuses through the cell
membrane. The unoccupied steroid recep-
tor (R) is coupled to a molecular chaperone
(HSP90) that stabilizes R in the correct
shape. When the hormone binds to the
receptor-HSP complex, the HSP dissociates
and the remaining hormone-receptor
complex dimerizes before it enters the cell
nucleus. The hormone-receptor dimer then
binds to target genes via a specific HRE.
Various factors such as GTFs and RNA POL II
assist in inducing gene transcription and
the export of mRNA into the cell cytosol
where it is translated into protein.
Abbreviations: GTF, general transcription
factor; HSP, heat shock protein; HSP90, heat
shock protein 90; mRNA, messenger RNA;
POL II, polymerase II; RNA, ribonucleic acid.
inflammation and osteoporosis (Paterni et al., 2014;
Warner et al., 2017).
Hormone Resistance
This section uses the estrogen and GC receptors as
examples of the clinical consequences of hormone
resistance. Other well-described examples include
androgen receptor insensitivity (testicular feminiza-
tion syndrome; Hiort, 2013) and thyroid hormone-
receptor insensitivity (Ortiga-Carvalho et al., 2014;
see also Chapter 6).
Estrogens
ER resistance appears to be unknown within the neu-
roendocrine system. However, mutations in ERα in
metastatic breast cancer are well-described and result
from long-term treatment with antiestrogens, such as
tamoxifen, and drugs such as fulvestrant that
degrades ERα (Huang et al., 2017). Drug-induced
estrogen resistance could take the form of changes in
downstream signaling or as intrinsic activation of ERα
in the absence of estradiol. It is possible that tamox-
ifen or fulvestrant could affect hypothalamic or pitui-
tary ERα receptors.
 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms
Glucocorticoids
GCs influence multiple physiological processes (see
Chapter 5), and are routinely used to treat disorders of
inflammation, autoimmune diseases and cancer.
The physiological effects of GCs can, in rare cases,
be compromised because of mutations in the GC
receptor that impair GC action. This reduces tissue
sensitivity to GCs, compromises negative feedback
and induces hypersecretion of ACTH, cortisol and
androgens (Figure 1.11; Charmandari et al., 2008;
Charmandari et al., 2013).
GC receptor sensitivity is also reduced when syn-
thetic GCs are used to suppress allergic, inflammatory
and immune disorders. Patients receiving chronic
treatment often develop GC insensitivity and
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Figure 1.9 Distribution of hERα and hERβ
throughout the body. Localization of hERα and
hERβ gene expression in normal human pituitary
(Chaidarun et al., 1998) and pituitary adenomas
(Manoranjan et al., 2010) has been added. Not
included in the figure is the localization of both
receptors in the human uterus (Simmen and
Kelley, 2016). Image reproduced with permission
(Warner et al., 2017). Abbreviations: hERα,
estrogen receptor α; hERβ, estrogen receptor β.
resistance, increasing patient vulnerability to exagger-
ated inflammatory responses. GC usage is increasing as
a result of disease prevalence in an aging population.
For example, GCs are used in the treatment of asthma,
allergic rhinitis, hematologic malignancies, ulcerative
colitis, rheumatoid arthritis, eczema and psychological
disorders. GC resistance can also occur as a result of
chronic stress as well as in major depression
(Rodriguez et al., 2016). Efforts to generate new, clini-
cally useful, GC receptor ligands that can overcome GC
resistance are in progress (Vandewalle et al., 2018).
1.5 Chapter Summary
The neuroendocrine hypothalamus influences endo-
9
crine targets in tandem with the pituitary gland; that
 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

Central nervous system Skin, mucosal and hair changes
• Vasomotor symptoms • Reduced skin thickness
• Sleep disruption • Reduced elasticity
• Depression and anxiety • Reduced hydration
• Cognitive changes • Increased wrinkling
• Migraine • Hair loss
Weight and metabolic changes
• Weight gain
• Increased visceral adiposity
Sexual function • Increased waist circumference
• Decreased sexual desire
• Dyspareunia
Figure 1.10 Effects of estrogen depletion in menopause.
Symptoms of menopause include CNS-related disorders,
weight gain, bodily alterations related to cardio-
metabolic changes, musculoskeletal alterations,
urogenital and skin atrophy and sexual dysfunction.
Perimenopause is associated with the worst menopausal
symptom burden, arising from neurochemical changes
within the CNS leading to severe vasomotor symptoms,
sleep disorders and depression, which might affect
cognitive function. Reproduced with permission
(Monteleone et al., 2018). Abbreviation: CNS, central
nervous system.

Urogenital system
• Vaginal dryness
• Vulvar itching and burning
• Dysuria
• Urinary frequency
• Urgency
Musculoskeletal system
• Recurrent lower urinary
• Joint pain
tract infections
• Sarcopenia

Figure 1.11 Changes in the HPA axis due to glucocorticoid resistance.

Glucocorticoid negative feedback at the hypothalamic and anterior pituitary
levels is compromised because of glucocorticoid resistance. This results in
increased secretion of CRH and ACTH, followed by adrenal hyperplasia, and
increased secretion of adrenal cortisol and androgens. Abbreviations: HPA,
hypothalamic–pituitary–adrenal. Figure derived from Charmandari et al. (2008).

10

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 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms
is, pituitary hormone secretion is directed by stimuli
secreted from hypothalamic neurons. The target
organ sensitivity to stimulation, and the neuronal
response to hormonal feedback, is dependent upon
a variety of receptor mechanisms.
Peptide hormones (e.g., oxytocin), neuropeptides
(e.g., thyrotropin releasing hormone) and neurotrans-
mitters (e.g., dopamine) regulate cellular activity by
binding to specific receptors located in target cell
membranes. Biochemical changes within the target
cell are induced via intracellular second messengers,
such as cAMP and protein kinase C. The transduction
of information from the membrane to the second
messenger is accomplished through the activation of
membrane proteins (e.g., G proteins) and enzymes,
such as adenylate cyclase. GPCRs are characterized by
their seven transmembrane domain structures and are
attached to trimeric G proteins. There are stimulatory
(Gs) and inhibitory (Gi) proteins. For example, GH
secretion is inhibited by somatostatin (Gi) and stimu-
lated by GHRH (Gs). The responsiveness of GPCRs is
compromised in some neuroendocrine disorders. For
example: (a) when GPCRs are subjected to chronic
stimulation to produce tachyphylaxis, and (b) when
mutations occur in the genes for receptor proteins or
G proteins; that is, loss-of-function or gain-of-
function mutations.
A second type of membrane stimulation is via
tyrosine kinase-dependent receptors. Peptide hor-
mones such as leptin, PRL and GH bind to receptors
having only two transmembrane domains and which
activate tyrosine kinase signaling, employing
the second messenger JAK/STAT pathway. The clin-
ical consequences of abnormal receptor signaling are
profound. For example, obese individuals are insensi-
tive to their own leptin (“leptin resistance”) and high
levels of endogenous leptin, derived from the
increased fat mass, fail to reduce food intake or body
weight. Patients with mutations in the leptin receptor
are also severely obese. Loss-of-function mutations in
the GH receptor result in severe growth failure and
IGF-1 deficiency. GH insensitivity is also caused by
mutations in the intracellular STAT5 gene. Loss-of-
function mutations in the PRL receptor present with
hyperprolactinemia, possibly due to the inability of
PRL to exert negative feedback on pituitary
lactotrophs.
In contrast, steroid hormones – such as estradiol
and cortisol – exert their effects via intracellular
receptors that have common structural elements
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and belong to a superfamily of receptor proteins.
The receptor proteins have no biological activity
until they bind to a hormone; that is, the hormone-
receptor complex acts as a transcription factor at
specific sites (HRE) on target genes. Two ERs (ERα
and ERβ) are encoded by two distinct genes and are
localized in many tissues, including the hypothala-
mus and anterior pituitary. These sites are critical for
regulation of the menstrual cycle. GC receptors are
also localized to the hypothalamic–pituitary system
where they control ACTH secretion. The physiolo-
gical effects of GC can be compromised because of
mutations in the GC receptor that impair GC action.
This reduces tissue sensitivity to GCs, compromises
negative feedback and induces hypersecretion of
ACTH, cortisol and androgens. GC resistance can
also occur as a result of chronic stress. Other well-
described examples of hormone resistance include
androgen-receptor insensitivity (testicular feminiza-
tion syndrome) and thyroid hormone-receptor
insensitivity.
1.6 Review Questions
1. Cyclic adenosine monophosphate is a well-
described second messenger involved in the
activation of many cell types. Name two
other second messenger systems.
2. Neuropeptides and neurotransmitters bind to
receptors located in the cell membrane. Based on
structure alone, there are two major membrane
receptor types. What are they?
3. Name five hormones that act through membrane
receptors.
4. Which of the following signaling molecules act via
a cytosolic receptor, rather than a membrane
protein?
a. Insulin-like growth factor 1
b. Cortisol
c. Growth hormone (GH)
d. Thyroxine (T4)
e. Somatostatin
5. The target cells of a hormone such as cortisol
respond to stimulation because:
a. the genome contains hormone response
elements
b. cortisol receptors are present in the cell
membrane
c. only target cells contain cortisol receptors 11
 Chapter 1: Basic Principles in Clinical Neuroendocrinology I: Receptor Mechanisms

d. cortisol receptor dimers are formed Brooks A J & Waters M J. (2010). The growth hormone
e. cortisol stimulates G protein-coupled receptors receptor: mechanism of activation and clinical implications.
Nat Rev Endocr 6, 515–525.
6. “Intracellular steroid hormone receptors have no
intrinsic biological activity.” Is this statement true Chaidarun S S, Swearingen B & Alexander JM. (1998).
Differential expression of estrogen receptor-β (ERβ) in human
or false? pituitary tumors: functional interactions with ERα and
7. The somatotroph contains three distinct signaling a tumor-specific splice variant. J Clin Endocr Metab 83,
mechanisms that regulate GH secretion; that is, 3308–3315.
GH releasing hormone (GHRH), somatostatin Charmandari E, Kino T, Ichijo T & Chrousos G P. (2008).
and ghrelin combine to control GH release. Which Generalized glucocorticoid resistance: clinical aspects,
G proteins are involved? molecular mechanisms, and implications of a rare genetic
8. Loss-of-function mutations in the genes for disorder. J Clin Endocr Metab 93, 1563–1572.
receptor proteins reveal their importance in Charmandari E, Kino T & Chrousos G P. (2013). Primary
endocrine/neuroendocrine disorders. Which generalized familial and sporadic glucocorticoid resistance
disorders are associated with mutations in the (Chrousos syndrome) and hypersensitivity. Endocr Dev 24,
following receptors? 67–85.
a. Kisspeptin receptor Dodington D W, Desai H R & Woo M. (2018). JAK/STAT –
Emerging players in metabolism. Trends Endocr Metab 29,
b. Thyrotropin releasing hormone receptor
55–65.
c. Melanocortin 4 receptor
d. GHRH receptor Engel J B & Schally A V. (2007). Drug insight: clinical use of
agonists and antagonists of luteinizing-hormone-releasing
e. Vasopressin receptor
hormone. Nat Clin Practice Endocr Metab 3, 157–167.
9. Which of the following hormonal patterns is Farooqi IS, Wangensteen T, Collins S et al. (2007). Clinical
associated with male precocious puberty due to an and molecular genetic spectrum of congenital deficiency of
activating luteinizing hormone (LH) receptor the leptin receptor. New Engl J Med 356, 237–247.
mutation? Fukami M, Suzuki E, Igarashi M, Miyado M & Ogata T.
a. Low testosterone, low LH and follicle (2018). Gain-of-function mutations in G-protein–coupled
stimulating hormone (FSH) receptor genes associated with human endocrine disorders.
b. Normal testosterone, high LH and FSH Clin Endocr 88, 351–359.
c. High testosterone, low LH and FSH Hauser A S, Attwood M M, Rask-Andersen M, Schiöth H B
d. Low testosterone, high LH and FSH & Gloriam D E. (2017). Trends in GPCR drug discovery:
new agents, targets and indications. Nat Revs Drug Discovery
e. Normal testosterone and LH and FSH
16, 829–842.
10. An activating mutation of the thyroid stimulating
Hiort O. (2013). Clinical and molecular aspects of androgen
hormone (TSH) receptor is associated with which insensitivity. Endocr Develop 24, 33–40.
of the following?
Huang D, Yang F, Wang Y & Guan X. (2017). Mechanisms of
a. High TSH resistance to selective estrogen receptor down-regulator in
b. High T4 metastatic breast cancer. Biochim Biophys Acta 1868, 148–156.
c. Goiter Hwa V. (2016). STAT5B deficiency: impacts on human
d. Features of hyperthyroidism growth and immunity. Growth Horm IGF Res 28, 16–20.
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13
 Chapter
Basic Principles in Clinical
2 Neuroendocrinology II:
Assays, Rhythms and Pulses
The clinical investigation of neuroendocrine pro-
blems is dependent on the quantitative assay of
hormone levels, for example, by immunoassay or
mass spectrometry. Blood samples are the primary
source for quantification of hormone concentra-
tions, although saliva and hair samples can also
provide valuable information. Automated blood
collection and automated assays permit the deter-
mination of circadian and rapid, pulsatile changes
in, for example, pituitary hormone secretion. This
chapter will outline the methodology and applica-
tion of these assay techniques.
2.1 Determination of Hormone Levels
The levels of circulating hormones can be determined
directly in blood samples or, in the case of steroid
hormones such as cortisol, in the saliva or hair.
The determination of glucocorticoid (GC) levels in
hair, for example, is able to detect prolonged exposure
to stress (Wester and van Rossum, 2015). This section
will include two routine techniques for the determi-
nation of hormone levels: (a) immunoassay and (b)
mass spectrometry.
Immunoassays
Since the 1960s the benchmark in determination of
hormone levels was the radioimmunoassay. However,
this method, employing antibodies specific to each
hormone, and radioactively labeled hormones, is
slow, labor intensive and raises safety problems in
the use and disposal of radioactive materials.
The ready availability of highly specific monoclonal
antibodies for steroid and peptide hormones aided the
development of rapid and automated chemilumines-
cent or immunometric assays that produce data in
a matter of hours, rather than days. A widely used
assay is the ELISA. One form of this technique (there
are several variations; Aydin, 2015) for the quantifica-
14 tion of cortisol levels in blood is illustrated in
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Figure 2.1. To perform this assay, a specific cortisol
monoclonal antibody (the “capture” antibody) is per-
manently attached to the bottom of a plastic micro-
well plate well (Step A). Cortisol samples are then
added and allowed to complex with the immobilized
antibody (Step B). Unbound products are then
removed with a wash step, leaving cortisol bound to
the antibodies. A biotin-labeled second antibody, also
specific for cortisol (the “detection” antibody), is then
added to the wells, binding to the captured cortisol
(Step C). After a further wash step, the biotinylated
detection antibody is itself now labeled with an
enzyme conjugate capable of generating light or fluor-
escence in response to a particular substrate (Step D).
The quantification step measures the intensity of light
produced after the addition of the substrate (Step E).
Standard curves are generated by adding known
quantities of cortisol, and these curves then permit
determination of unknowns in the blood samples.
The availability of monoclonal antibodies for
many hormones and peptides is a major advantage
of this method. In general, the samples do not need to
be purified prior to use, and the whole process can be
automated to handle large numbers of samples.
Typical uses of this technique are: (a) estimation of
pulsatile secretion of human adrenocorticotropin
(ACTH) and cortisol (Henley et al., 2009; cf.
Chapter 5, Figure 5.3), and (b) the determination of
salivary cortisol (Langelaan et al., 2018).
A disadvantage of this technique is the inability to
assay more than one hormone in a single clinical
patient sample. Complex pathologies may require
determination of a panel of hormones for diagnosis
or prognosis. Automated multiplex immunoassay
technologies are increasingly available, and
Figure 2.2 outlines just such a method for the simul-
taneous assay of insulin, growth hormone (GH), lep-
tin, thyroid stimulating hormone (TSH) and
glucagon-like peptide-1 (GLP-1) (Stephen and
Guest, 2017).
 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Addition of Addition of
enzyme chemiluminescent substrate
complex
Chemiluminescent
Biotin-labeled substrate
HRP
detection conjugate
antibody
Light
B B B

Cortisol
Cortisol-specific binding
monoclonal
antibody

A B C D E
Figure 2.1 Assay of cortisol using ELISA. Each well of a microwell plate has been pre-coated with a cortisol-specific monoclonal antibody
(capture antibody) (A). Standard amounts of cortisol, or samples, are added to the wells and cortisol binds to the capture antibody (B).
Unbound standard or sample is washed away. A biotin-conjugated, cortisol-specific, detection antibody is then added, which binds to the
captured cortisol (C). Unbound detection antibody is washed away. An avidin–HRP conjugate is then added, which labels the biotin (D).
Unbound avidin–HRP conjugate is washed away. Addition of a chemiluminescent substrate causes the HRP enzyme to induce light emission
(E). The light units of each well are automatically measured. Light units from unknown samples can then be compared with a light unit
standard curve generated using known cortisol concentrations in order to determine serum cortisol concentrations. See www.lsbio.com/pr
oducts/elisakits/clia as a typical example of this technique. Abbreviations: HRP, horseradish peroxidase.

Specific antibodies are covalently attached to dis-
tinct, dye-coded microspheres and added to the hor-
mone sample. Each hormone binds to the appropriate
antibody (simplified here by only showing the
sequence for GH). As in Figure 2.1, the hormone-
bound antibody is further decorated with a fluorescent
biotin conjugate. The complete mixture – micro-
spheres, hormones and decorated antibodies – is sepa-
rated into antibody-specific groups by flow cytometry
and then analyzed by lasers able to quantify each dye-
coded microsphere as well as the fluorescence of the
bound hormone. This fully automated technique
enables much information to be derived from a single
sample, with minimal reagent usage, at lower cost
compared with the assay shown in Figure 2.1.
Mass Spectrometry
A second powerful technique to quantify multiple
hormones in single samples of biological fluids is
mass spectrometry. The biggest advantage it possesses
over the immunoassays is that it does not require
antibodies to assay the hormones of interest. This is
not to say that the ELISA is not useful; there are many
hormones for which highly specific antibodies are
available and therefore can be quantified with
ELISAs. However, even the most specific antibodies
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may still cross-react with other hormones and this
lack of specificity produces misleading results.
In contrast, the mass spectrometer can determine
absolute values of many substances in blood, with
high specificity, based on differences in molecular
weight. High throughput testing is possible, using
small sample volumes with minimal sample prepara-
tion, and eliminating the requirement for expensive
immunoassay-specific reagents such as antibodies.
For example, when coupled with liquid chromatogra-
phy to separate sample components, mass spectro-
metry is an efficient and rapid way to assay steroid
hormones in a clinical setting (Matysik and Liebisch,
2017). This technique may also be the only way to
determine very low levels of hormones in blood; for
example, estradiol levels in prepubertal children
(Stanczyk and Clarke, 2014) or low levels of cortisol
in saliva (Sturmer et al., 2018).
The collection of biological samples such as saliva,
hair and urine for the analysis of hormone levels is
simple, non-invasive and stress-free compared with
taking blood samples. Hormone levels can therefore
be determined in newborn children, as well as adults,
outside the laboratory or hospital setting, and mass
spectrometry permits the analysis of multiple hor-
15
mones simultaneously in the same sample. Also,
 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Figure 2.2 Overview of the multiplex immunoassay protocol. Samples are added to dye-coded microsphere–antibody complexes that capture
specific hormone targets. The capture of GH is shown in this sequence. Following incubation with a fluorescent biotinylated label, the
mixtures are streamed through the detector – via flow cytometry to separate the individual groups of microspheres – which uses lasers for
identification of the antibody–microsphere conjugates and quantitation of the fluorescent bound hormones. The example shows a 5-plex
assay capable of binding the targets GLP-1, GH, insulin, leptin and TSH. This figure is based on the illustration from Stephen et al. (2017).

since cortisol is deposited in the growing hair shaft, an
estimate of cortisol concentrations in hair serves as
a measure of chronic exposure over weeks and months
(Meyer and Novak, 2012; Wester and van Rossum,
2015). Nonetheless, it is important to remember that
hair, saliva and urine analysis only provides a measure
of the average hormone secretion over a number of
hours or weeks. In the case of cortisol, for example,
blood samples taken every few minutes is the only way
to demonstrate pulsatile secretion and its circadian
variation (see Figure 5.3 showing pulsatile secretion
of ACTH and cortisol). Table 2.1 summarizes the
variations in hair cortisol levels associated with some
clinical situations (Wester and van Rossum, 2015).
2.2 Patterns of Hormone Secretion
The neuroendocrine system is defined by rhythms
16 that orchestrate the dialogue between the brain and
critical systems such as regulation of growth, repro-
duction and stress. Some of these rhythms have per-
iods longer than 24 hours, typified by the female
menstrual cycle. Circadian or 24-hour rhythms
include the sleep–wake cycle and the increase, for
example, in GH secretion seen at night. There are
also cycles of less than 24 hours – the ultradian
cycles – such as the pulsatile release of luteinizing
hormone (LH), follicle stimulating hormone (FSH)
and GH.
Section 2.1 outlined the analytical approaches
used to assess hormone levels in biological fluids.
Since hormone levels often vary dramatically through
24 hours, it is critical to determine hormone secretory
patterns in normal individuals to inform when hor-
mone samples should be obtained in clinical practice.
The following sections outline the clinical importance
of normal hormonal rhythms.

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 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Table 2.1 Clinical and situational factors associated with variations in hair cortisol levels

Increased hair cortisol Decreased hair cortisol

Somatic health factors
Cushing’s syndrome Childhood asthma with inhalation
glucocorticoids
Hydrocortisone use
Obesity
Metabolic syndrome
Diabetes mellitus
Cardiovascular disease
Heart failure severity
Recent myocardial infarction
Chronic and acute stressors
Intensive aerobic exercise Traumatic experience
Trauma
Life events
Unemployment
Shift work
Severe chronic pain
Psychopathology
PTSDa PTSDa
Major depressive disorder Generalized anxiety disorder
Bipolar disorder, late onset Panic disorder
a
Post-traumatic stress disorder (PTSD) has been associated with both increased and decreased hair cortisol concentrations (depending on
the type of traumatic event, characteristics of the patient sample examined, and the timespan between the trauma and assessment)
compared with controls (Wester and van Rossum, 2015).

and peak secretion for these is illustrated in

Daily Rhythms
As an example of a well-described daily rhythm,
Figure 2.3 illustrates that pulsatile cortisol levels in
healthy individuals vary with a 24-hour period, with
blood levels beginning to rise late in the night to peak at
the sleep–wake transition (cf. Chapter 5; Figure 5.3).
The figure also reveals that in patients with pituitary-
dependent Cushing’s disease, although the frequency
of cortisol pulses is increased, producing significant
cortisol excess, the diurnal pattern of cortisol output
is absent (van den Berg et al., 1995). These data indicate
dysfunction of the hypothalamic–pituitary system in
Cushing’s disease. These rhythms may also be dis-
rupted in other disease states. For example, psychotic
disorders such as major depression are associated with
changes in circadian rhythms similar to Cushing’s syn-
drome (Keller et al., 2006).
Levels of several other circulating endocrine
factors are known to oscillate through 24 hours,
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Figure 2.4.
The secretory rhythms of some hormones (e.g.,
cortisol and TSH) occur independently of sleep-
wake cycles. In a study on healthy young men
the cortisol profile was minimally affected by the
absence of sleep or when they slept at an abnormal
time of day. Over a period of 28 hours of continuous
wakefulness, constant light and constant caloric
intake, the normal wave shape of the rhythm of corti-
sol release was maintained (Oster et al., 2017). This
finding indicates that fluctuations in the levels of some
hormones, over the course of the day, are driven, at
least in part, by an endogenous circadian mechanism
(i.e., independent of behavioral rhythms). In contrast,
24-hour oscillations in the levels of other endocrine
factors (e.g., GH and prolactin) are dependent on
sleep–wake cycles. For example, sleep deprivation
abolishes GH secretion, but daytime sleep, several 17
hours later, restores the sleep-related surge of GH
 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Figure 2.3 Diurnal rhythm of human cortisol secretion: control vs.

Cushing’s disease. Circulating cortisol concentrations in a control male
Cushing’s disease (lower profile) and a male patient with Cushing’s disease (upper
1500
profile). Note that both profiles demonstrate the presence of pulsatile
secretion of cortisol, but the 24-hour variation is absent from the
Cushing’s patient. Data obtained from van den Berg et al. (1995).
Cortisol (nmol/L)

1000

800

Lights
600
off
400

200
Control

0
0900 1200 1500 1800 2100 2400 0300 0600 0900
Clock time

Figure 2.4 Peak

variations in circulating
endocrine factors.
The time of day at which
circulating levels of key
endocrine factors peak in
humans. Abbreviations:
FGF 21, fibroblast growth
factor 21; RAAS,
renin–angiotensin–
aldosterone system; T3,
triiodothyronine. Image
reproduced with permis-
sion (Gamble et al., 2014).

(van Cauter et al., 1998; cf. Figure 8.2). Seminal stu- adrenal, liver and adipose tissue – also contain sec-
dies in experimental animals revealed that a specific ondary clock mechanisms that synchronize with the
structure in the hypothalamus – the suprachiasmatic SCN master oscillator (Figure 2.5; reviewed in Oster
nucleus (SCN) – is the site of the clock mechanism et al., 2017).
that governs circadian variations in hormone release, The molecular circuitry that governs the produc-
as well as a variety of physiological functions. Most tion of rhythms, perhaps common to all these oscilla-
18 peripheral tissues, throughout the body – including tors, is represented schematically in Figure 2.6. In its
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 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Pineal gland Figure 2.5 Schematic representation of

synchronization of SCN clock with periph-
eral oscillators. Both neural signals
(transmitted by the autonomic nervous
system; black arrows) and hormonal
signals are involved. The 24-hour rhythms
Plasma melatonin SCN of circulating melatonin (released by the
pineal gland) and cortisol (from the
adrenal cortex) are considered as primarily
controlled by the central SCN clock.
The blue and purple arrows symbolize,
Plasma GCs respectively, the synchronizing effects of
sleep
the GC and melatonin rhythms. Due to the
0 12 24 ubiquity of glucocorticoid receptors in the
Time of day (h) entire organism, the 24-hour rhythm of
circulating GCs plays a major role in
sleep
synchronizing central and peripheral
clocks. Reproduced through open access
0 12 24 (Oster et al., 2017).
Time of day (h)

Adrenal
gland

Figure 2.6 Schematic outline

of how clock gene expression in
BMAL1 RHYTHMIC the SCN regulates circadian
SIGNALS rhythms. Positive and negative
CLOCK gene expression loops
produce output signals
responsible for rhythmic cell
+ function over 24 hours.
The clock genes bmal1 and
clock encode the proteins
bmal1 mRNA BMAL1 and CLOCK that form
clock mRNA a heterodimer transcription
factor that stimulates the per
and cry genes. In turn, their
protein products, CRY and
bmal1

clock per cry PER, reach a critical level

before negatively regulating
the activity of bmal1 and clock,
thereby reducing the amount
NUCLEUS cry mRNA
of CLOCK and BMAL1. Image
per mRNA of DNA structure reproduced
with permission: https://com
mons.wikimedia.org/w/index
.php?title=File:DNA_structur
– e_and_bases_color_FR.svg
CRY &oldid=124826534.

PER
SLEEP,
CIRCADIAN INFORMATION CYTOPLASM
HORMONES,
STRESS, etc.
19

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 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses
simplest form, neurons in the SCN begin to produce
two proteins at the start of the circadian day. These
proteins – BMAL1 and CLOCK – form a heterodimer
that acts as a transcription factor able to stimulate
expression of two further genes – per and cry – that
encode the proteins PER and CRY. The buildup of
PER and CRY over the course of the day establishes an
autoregulatory feedback loop that represses the clock
and bmal1 genes, reducing the levels of CLOCK and
BMAL1. This feedback cycle takes approximately 24
hours, driving the circadian rhythm. For example, the
circadian signal necessary for the production of night-
time cortisol (Figure 2.3) would be an increase in
corticotropin releasing hormone (CRH) neuron activ-
ity, increasing CRH secretion into the pituitary portal
system (see Figure 5.2).
Current evidence suggests that the human circa-
dian system also uses clock genes. The human SCN
expresses the clock gene (Steeves et al., 1999), and
circadian variations in clock mRNA and bmal1
mRNA have been detected in human placenta (Pérez
et al., 2015). A clock/bmal1 mechanism may therefore
be responsible for human circadian rhythms, includ-
ing variable adrenal sensitivity to stimulation by
ACTH (Angelousi et al., 2018). In addition, poly-
morphisms in clock genes are implicated in the sus-
ceptibility to obesity (Gómez-Abellán et al., 2008;
Sookoian et al., 2008). For example, per2 expression
in adipose tissue was negatively correlated with waist
Table 2.2 Human circadian rhythm disruption
Source
Behavioral
Light Meal/fasting
cycle cycle
disruption
Work schedules (e.g., shift work) ✓ ✓
Jet lag ✓ ✓
Unusual photoperiods (e.g., polar ✓ ✗
regions)
Circadian rhythm sleep/wake ✓ ✗
disorders (e.g., non-24-hour
sleep/wake disorder)
Aging ✗ ✗
Disease states (e.g., Alzheimer’s) ✗ ✗
20 Data derived from Potter et al. (2016)
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circumference in morbidly obese patients (Gómez-
Abellán et al., 2008), and a clock polymorphism (in
white blood samples) was associated with a 1.8-fold
risk of obesity (Sookoian et al., 2008).
Neuroendocrine Consequences of Circadian
Disruption
Human circadian rhythms are especially sensitive to
multiple adverse influences; for example, shift work,
artificial lighting at night and long-distance flights
across time zones (jet lag). The evidence is clear that
chronic perturbation of circadian function provokes
metabolic, reproductive, sleep and mood disorders
(Sheikh-Ali and Maharaj, 2014; Bedrosian et al.,
2016; Nicolaides et al., 2017). Some of these issues
are summarized in Table 2.2 (Potter et al., 2016).
The light-dependent rhythm of melatonin secre-
tion from the pineal gland (see Figure 2.5) is
a sensitive indicator of circadian disruption. For
example, low light levels in the early night phase are
sufficient to delay the melatonin rhythm, and the use
of a computer or e-book before bed depresses mela-
tonin secretion and may disrupt sleep (Bedrosian
et al., 2016; Potter et al., 2016). Night-shift workers
are exposed to light levels that far exceed those that
affect the circadian system. Several studies of shift
workers indicate that exposure to nighttime light
leads to metabolic problems. For example, healthcare
Mechanism
Biological
Rest/activity Genetic Physiological
disruption disruption disruption (e.g.,
(e.g., clock retinal dysfunction)
gene
mutations)
✓ ✗ ✗
✓ ✗ ✗
✗ ✗ ✗
✗ ✓ ✓
✗ ✗ ✓
✗ ✓ ✓
 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses

Figure 2.7 Exposure to night-

US light pollution US obesity trends time light parallels incidence of
obesity. The left panel shows
light pollution trends in the
United States from the mid-
1970s through projected levels
in 2025. The right panel shows
obesity trends in the United
States from 1990, 2000 and
2010. Reproduced with per-
mission (Fonken and Nelson,
2014). Abbreviation: BMI, body
mass index.
mid-1970s
1990

1977

2000

2025
<11% above the natural brightness level
11–33% above the natural brightness level
34–99% above the natural brightness level
100% above the natural brightness level 2010
3–9 times the natural brightness level
(the Milky Way is no longer visible) No data Obesity is defined as BMI
<10%
9–27 times the natural brightness level
10–14% ≥30, OR ~30 Ibs
(fewer than 100 stars are visible) overweight for a
15–19%
27–81 times the natural brightness level 20–24% 5′4′′ person
(the North Star is no longer visible) 25–29%
81–243 times the natural brightness level ≥30%
(the Big Dipper is no longer visible)

personnel that work night shifts appear to be at risk of
developing metabolic syndrome; that is, in men and
women, increases in hypertension and cholesterol, obe-
sity, and hypertriglyceridemia (Fonken and Nelson,
2014; Sheikh-Ali and Maharaj, 2014). This effect
could be due to a combination of exposure to nighttime
light plus the behavioral de-synchrony of working at
night, although a marked elevation in exposure to light
at night in the US general population has paralleled the
increased incidence of obesity (Figure 2.7).
Aging is also associated with circadian disruption,
especially in sleep–wakefulness and hormonal
rhythms. The circadian pacemaker in the human
brain becomes progressively disturbed through neu-
ronal degeneration of the SCN, and this is especially
notable in Alzheimer’s disease (Hofman and Swaab,
2006). With respect to hormonal changes, a study on
age-related changes in sleep and GH secretion in men
aged 16–83 years showed that the amount of slow
wave sleep decreased by 80% between the young
(16–25 year olds) and middle-aged (36–50 year olds)
(Van Cauter et al., 2000). This was associated with
a 75% reduction in spontaneous GH secretion
(Blackman, 2000). Small increases in early-night cor-
tisol have also been detected in healthy old adults
(Vgontzas et al., 2003). Disruption of the rhythm of
circulating GCs impacts multiple functions, including
energy metabolism, stress, immune function and cog-
nition (Oster et al., 2017; cf. Chapter 5). Abnormal
rhythms of cortisol are linked with shift work, 21

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 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses
Cushing’s syndrome, adrenal insufficiency, anxiety and
depression, and PTSD. For example, chronic stress
abolishes the cortisol circadian rhythm and increases
appetite, obesity, and metabolic disturbances, such as
hyperglycemia, insulin resistance, dyslipidemia, osteo-
penia/osteoporosis and hypertension (Nader et al.,
2010). Women in the healthcare and transportation
industries are at a high risk of shift-work-associated
fertility problems, including abnormal reproductive
cycles, increased latency to pregnancy and miscarriages
(Sen and Sellix, 2016). In general, the abnormal cortisol
rhythm represents an abnormally high level in the
afternoon and evening. The normalization of the cir-
cadian rhythm of cortisol should therefore target the
evening to re-establish the physiological low point of
cortisol secretion. Pharmacological suppression of cor-
tisol biosynthesis has been used in healthy human
adults to elucidate metabolic implications of cortisol
rhythmicity (Oster et al., 2017). In a multicenter retro-
spective study, the synthesis inhibitor metyrapone was
an effective therapy for short- and long-term control of
hypercortisolemia (Daniel et al., 2015).
Pulsatile Secretion
In subsequent chapters, we will detail the pulsatile
pattern of human LH, ACTH, TSH, PRL, GH and
oxytocin secretion within their respective
hypothalamic–pituitary–target organ systems. It is
relevant that the phenomenon of episodic secretion
is not exclusive to pituitary hormones. For example,
insulin secretion from pancreatic islet β-cells is also
pulsatile, with a frequency of approximately 6–10
minutes in healthy adults (Schmitz et al., 2002).
Pulsatile stimulation of the anterior pituitary with
releasing hormones to increase secretion of circulat-
ing signaling molecules appears to be the optimal
mode of communication. In general, a continuous
stimulation of receptors, rather than a pulsatile sig-
nal, will lead to a decrease in responsiveness of the
target cell; that is, via receptor desensitization. Thus,
the time between pulses should be sufficient to
permit a receptor to re-sensitize.
In this section, we will describe two systems to
exemplify the importance of pulsatile secretion; that is,
gonadotropin releasing hormone (GnRH; reproductive
system) and CRH (hypothalamic–pituitary–adrenal sys-
tem; HPA). Using the human female reproductive sys-
tem as an example (described more fully in Chapter 3),
22 there is an optimal frequency of pituitary stimulation
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with GnRH that establishes LH and FSH levels that are
consistent with fertility (ovulation). The actual frequency
of GnRH pulses appears to be important in the normal
menstrual cycle. Figure 3.5 (in Chapter 3) reveals that
the frequency of pulses at midcycle is greatly enhanced
to induce a surge in LH, followed by ovulation.
In contrast, continuous stimulation of pituitary GnRH
receptors induces a state of receptor downregulation,
and loss of LH secretion, which is reversible by applying
pulses of GnRH. Figure 2.8 illustrates this principle in
a woman suffering from hypothalamic amenorrhea;
that is, there is no endogenous GnRH stimulation of
the pituitary, LH levels are low and ovulation does not
occur (Southworth et al., 1991). The figure shows that
continuous stimulation with GnRH fails to increase
secretion of LH, FSH and estradiol. In contrast, pulsatile
GnRH produces a sustained elevation in LH and FSH
levels, enabling a surge of estradiol secretion and
ovulation.
The clinical implications arising from these stu-
dies are two-fold: (a) pulsatile stimulation of pituitary
secretion of LH and FSH is mandatory for restoration
of gonadotropin secretion, ovarian estradiol produc-
tion and ovulation in the treatment of hypothalamic
amenorrhea; (b) the ability of continuous stimulation
with GnRH to suppress LH and estradiol to reduce
fertility. This latter effect is clinically advantageous in
various ways. For example, synthetic, long-acting
GnRH agonists induce a castration-like state, and
reduce gonadotropin and sex hormone levels in
advanced prostate cancer. They are also able to reduce
estradiol levels in the treatment of estrogen-sensitive
breast cancer, and are valuable in in vitro fertilization
protocols (Engel and Schally, 2007).
GnRH neurons appear to have an intrinsic ability
to secrete GnRH in an episodic manner, although
additional neural networks – impinging on GnRH
neurons – can modify pulse frequency and amplitude
(Millar, 2015). In contrast, the pulsatile secretion of
ACTH may not be the result of oscillation of hypotha-
lamic CRH neurons. ACTH pulses are generated
through a feedforward–feedback interaction between
CRH-stimulated pituitary corticotrophs and the cor-
tisol-producing cells of the adrenal cortex (Lightman,
2016; Figure 2.9). This mechanism shows some simi-
larities to the generation of circadian rhythms illu-
strated in Figure 2.6.
A tight correlation of high frequency ACTH and
cortisol pulses, through 24 hours in a healthy volun-
teer is shown in Figure 2.10 (Gibbison et al., 2015).
 Chapter 2: Basic Principles in Clinical Neuroendocrinology II: Assays, Rhythms and Pulses
The slight delay in cortisol pulses, following ACTH
stimulation, is due to the need for cortisol to be newly
synthesized before release. Each pulse of ACTH is
then inhibited by the rapid negative feedback of cor-
tisol exerted at the pituitary. This constitutes the
oscillatory rhythm.
This rapid response of the adrenal cortex to sti-
mulation with pulses of ACTH is therefore very dif-
ferent to that of the human ovary responding to LH
and FSH pulses. Figure 3.1 (Chapter 3) reveals that the
ovarian response to stimulation with gonadotropins is
not a series of estradiol pulses, but the release
a prolonged surge of estradiol over a 48-hour period.
There is evidence of abnormal cortisol rhythms in
depression, aging, increasing body mass index (BMI),
PTSD and obstructive sleep apnea (Lightman and
Terry, 2014). Equally problematic is the treatment of
GC deficiency, with GC-replacement regimens that
do not recapitulate the physiological pulsatile rhythm
of cortisol release. For example, patients undergoing
oral, non-pulsatile GC replacement in Addison’s dis-
ease (see Section 5.5) have double the mortality of age-
related controls as well as increased morbidity,
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Figure 2.8 Effects of pulsatile
vs. continuous stimulation with
GnRH. Hormone data
obtained every 1–3 days
throughout the continuous
and pulsatile infusions of
GnRH in a single patient with
hypothalamic amenorrhea.
Arrow indicates ovulation and
menses beginning on day 68
of the pulsatile regimen, but
not during the continuous
regimen. Data obtained from
Southworth et al. (1991).
especially related to mental and physical fatigue
(Lightman, 2016). Efforts to establish pulsatile levels
of cortisol with a novel programmable portable pul-
satile continuous subcutaneous delivery system have
been successful. Healthy volunteers, whose endogen-
ous secretion of cortisol was suppressed, responded
with a near-physiological pattern of circadian and
ultradian rhythmicity (Russell et al., 2014;
Figure 2.11).
In proof-of-principle studies, this technique was
used to demonstrate that the pattern of GC replace-
ment differentially modulates working memory and
sleep physiology. For example, abolition of physiolo-
gical cortisol pulsatility correlates with poorer work-
ing memory performance when cognitive demands
are high. In addition, disruption of ultradian rhyth-
micity results in a poorer self-perceived quality of
sleep (Kalafatakis et al., 2018). These authors con-
cluded that “Future studies in patients with adreno-
cortical insufficiency are now needed, not only to help
reduce the morbidity of current replacement regi-
mens, but also to provide evidence from longer-term
modification of replacement cortisol rhythmicity for 23
Another random document with
no related content on Scribd:
 2 ozs. butter
1 table-spoon white roux
¹⁄₂ salt-spoon carbonate of soda
Stew the tomatoes until very soft. Add the carbonate of soda and
sugar. Put through a fine sieve. Set in a small sauce-pan on the fire
to keep hot. Heat the milk, thicken it with white roux (flour and butter,
see p. 12). Let it boil a few minutes, stirring continually. Season. Add
the tomatoes and serve immediately. (The tomatoes should not be
added until actually ready to serve.)

Portuguese Soup
3 tomatoes
1 Spanish onion
A small bunch of herbs
2 large slices of stale bread
1 oz. grated cheese (parmesan)
1 quart hot water
1 oz. butter
Cut up the tomatoes and onions. Fry a light brown in butter. Put
them in a stew-pan and cover with a quart of hot water. Let it boil,
and then stand aside to simmer for half-an-hour. Strain off the liquid.
Rub the vegetables through a coarse sieve. Return to the fire,
season, and make very hot. Break up the bread and put it in the
bottom of a hot soup tureen. Sprinkle a little of the grated cheese
upon it. Pour the soup over it. Sprinkle the rest of the cheese on the
soup.

Potato Cream
1 pint milk
1 gill cream
2 potatoes
1 onion
1 tea-cup cooked French beans
 1 dessert-spoonful chopped cooked carrot
1 tea-spoon Liebig’s extract
1 small table-spoon white roux
Boil the potatoes and onion. Put them through a sieve. Add them
to the milk, which should be boiling. Add the white roux (see p. 12),
the Liebig (diluted with a little water) and seasoning. Stir for a minute
or two. Cut the French beans into small pieces. Add them and the
very finely chopped carrot to the soup. Stir in the scalded cream.

Potato Soup
3 potatoes
1 quart milk
1 table-spoon chopped onion
A little celery or ¹⁄₂ a tea-spoon celery salt
1 table-spoon white roux
1 table-spoon chopped parsley
Peel the potatoes. Soak them in cold water for half-an-hour. Cook
them in boiling water until soft. Drain off the water. Put the potatoes
through a sieve. Boil the milk with the onion and celery (or celery
salt). Strain. Add to the potatoes. Stir in the white roux (see p. 12).
Season. Boil for five minutes. Add the parsley.

Sorrel Soup
1 handful of sorrel
1 pint of water
1 tea-cup cream or milk
Bread
Wash and prepare a handful of sorrel. Put it in a sauce-pan with
the butter and a pint of water. Season. Boil gently for a quarter of an
hour. Add a little cream or milk. Put several very thin slices of bread
in the soup tureen, and pour the soup over them.
Rice or tapioca can be added to the soup.
 Summer Soup
1 cucumber
2 cabbage lettuces
1 onion
Small handful of spinach
A piece of mint
A pint of shelled peas
2 ozs. butter
A slice of ham
Wash the lettuces and cut them up. Cut up the cucumber and
onion. Put them with half a pint of peas, the mint, ham and butter into
a stew-pan. Cover with a little more than a quart of cold water. Bring
to a boil, and then simmer gently for three hours. Strain off the liquid.
Pass the vegetables through a sieve. Add to the liquid. Set on the
fire again. Season. Add half a pint of green peas which have already
been boiled.

Tomato Soup
1 tin of tomatoes
1 pint boiling water
1 table-spoon sugar
4 cloves
2 pepper-corns
1 table-spoon butter
1 ” flour
1 ” chopped onion
1 ” ” parsley
Put the tomatoes, water, sugar, cloves and pepper-corns in a
porcelain-lined sauce-pot. Simmer for half-an-hour. Fry the onions
and parsley in the butter, being careful not to burn. Add the flour to
them, mix smooth. Add them to the tomatoes. Simmer for ten
minutes. Strain through a fine sieve. Season. Serve with rice (see p.
106) or croûtons (see p. 103).
 Soups thickened with a Liaison of
Cream and Yolk of Egg
PAGE
Brown Bread Soup 51
Cauliflower Cream 51
Cream of Rice 52
Cucumber Soup 52
Dutch Soup 53
Flemish Soup 53
Friar’s Chicken 54
Italian Macaroni Soup—I 54
Macaroni Soup—II 55
Russian Soup 56
Turkish Soup 56
Water-cress Soup 57
White Chicken Soup 57
White Veal Soup 58
In thickening soups with a liaison of cream (or milk) and yolk of
egg, the eggs must first be well beaten, then the cream should be
added to them and thoroughly mixed. When this is done take a tea-
cup of hot stock and mix it slowly with the liaison. Strain it all through
a fine sieve or muslin, and add gradually to the soup, which must on
no account be allowed to boil after the liaison is added, although it
should be stirred over a gentle fire until it thickens.
In thickening soup with eggs only, beat the required number of
eggs, add a little warm stock to them. Strain, and add gradually to
the soup.

Brown Bread Soup

1 quart stock
4 or 5 small slices of brown bread
¹⁄₂ head of celery
1 carrot
4 table-spoons glaze
4 yolks of eggs
1 gill sour cream
Toast the brown bread. Add it with the sliced celery and carrot to a
rich stock from which all the fat has not been removed. Bring to a
boil. Simmer for an hour. Add four table-spoons glaze. Put all
through a sieve. Heat gently. Add the liaison of eggs and cream.
Serve with croûtons made of brown bread (see p. 103).
This soup can be made with German black bread.

Cauliflower Cream
1 quart chicken stock
1 cup cooked cauliflower
2 yolks of eggs
¹⁄₂ pint cream
1 cup button mushrooms
Put the cauliflower through a fine sieve. Add it to the boiling stock.
Season. Add the liaison of cream and egg.
Place the cooked mushrooms at the bottom of a soup tureen. Pour
the soup over them.

Cream of Rice with Parmesan

 ¹⁄₄ lb. Carolina rice
1 quart chicken stock
1 gill cream
¹⁄₂ oz. Parmesan
2 yolks of eggs
Wash the rice. Boil it for ten minutes in water. Drain. Add to the
stock. Simmer until the rice is tender. Put through a fine sieve. Add
to the stock. Mix in the cheese. Add the liaison of cream and eggs
(see p. 50).
Quenelles of chicken (see p. 105) can be added if desired, or rice
balls (see p. 107).

Cucumber Soup
1 cucumber
1¹⁄₂ pints white stock
1 oz. butter
1 onion
Small handful of sorrel
A little chervil
1 gill of cream
2 eggs
Cut the cucumber into thin slices. Sprinkle salt over them. Leave
them for an hour. Drain. Put them in a sauce-pan with the butter, the
onion, chervil and sorrel finely minced. Add the stock. Season.
Simmer for twenty minutes. Add the liaison of cream and eggs and
serve.

Dutch Soup
2 carrots
2 turnips
1 cucumber
1 quart chicken or veal broth
 Yolks of 3 eggs
1 gill of cream
Tea-spoonful butter
1 gill cooked French beans
1 gill cooked young peas
Cut the carrots, turnips and cucumber into olive-shaped pieces.
Blanch for three minutes in boiling water. Add to the stock, and
simmer until the vegetables are tender. Take off the fire. Season.
Add the yolks and cream and butter (in small pieces). Stir over the
fire until the soup thickens.
Put the freshly cooked peas and beans (cut into dice) into the
soup tureen. Pour the soup over them.

Flemish Soup
1 quart veal stock
1 handful spinach and sorrel
¹⁄₂ pint cream
3 yolks of eggs
Boil the chopped spinach and sorrel in the stock until tender.
Season. Just before serving add the liaison of eggs and cream. Stir
continually until it thickens. Serve with croûtons (p. 103).

Friar’s Chicken
1 quart veal stock
1 chicken
3 yolks of eggs
1 pint cream or milk
2 table-spoons chopped parsley
Cut the chicken into joints. Scald and skin them. Add them to the
stock. Season. Bring to the boil. Simmer gently for an hour. Skim
from time to time. Strain. Add the liaison of egg and cream, and the
parsley.
 Italian Macaroni Soup—I
5 ozs. macaroni
2 ozs. butter
1 quart white stock
¹⁄₂ pint cream or milk
3 yolks of eggs
1 oz. grated Parmesan
Cut the macaroni in boiling water, adding butter, salt and pepper.
Boil for half-an-hour. Drain. Cut in half-inch lengths.
Heat the white stock. Add the macaroni to it. Simmer another half-
hour. Add liaison of eggs and cream (or milk) and the grated cheese.

Macaroni Soup—II

¹⁄₂ lb. macaroni

A little more than 1 quart chicken stock
About 30 forcemeat balls
4 yolks of eggs
1 gill of cream
Boil the macaroni for ten minutes in cold water. Drain it. Cut it in
finger-lengths. Cook it again for fifteen minutes in a little clear
chicken stock. In a hot dish lay first a layer of macaroni, then one of
small chicken forcemeat balls (see p. 104), then another layer of
macaroni, etc.
Heat a quart of clear chicken stock to boiling point. Add a liaison of
the yolks of four eggs and a gill of cream. Strain. Serve in a soup
tureen with the dish of macaroni and forcemeat balls.

Russian Soup
2 kidneys
2 small onions
 ¹⁄₄ lb. mushrooms
1 dozen small olives
3 gherkins
1 quart strong stock
Yolks of two eggs
Melt the butter and fry the kidneys and onions (finely cut up) in it
very gently for five minutes. Cook the mushrooms separately. Put the
kidneys, onions, mushrooms, olives and gherkins (finely sliced) in a
hot soup tureen. Pour over them a quart of rich, dark, well-seasoned
brown stock, which has been thickened with the yolks of two eggs.

Turkish Soup
1 quart of veal or beef stock
¹⁄₂ a tea-cup of rice
2 yolks of eggs
1 table-spoon cream
Boil the rice and stock together until the rice is tender. Press
through a sieve. Season. Add the yolks and cream. Serve with
croûtons.

Water-cress Soup
3 potatoes
1 handful chopped water-cress
1 quart stock (or water)
2 yolks of eggs
1 table-spoon cream
1 oz. butter
1 tea-spoon white roux
Peel and wash the potatoes. Cook them in a little stock. When
tender mash them and put through a sieve. Add them to the rest of
the stock. Put back on the fire. Heat gently. Add a tea-spoonful of
white roux (see p. 12). Add the butter in small pieces.
 Make a liaison of the eggs and cream. Stir into the soup. Add the
water-cress uncooked. Serve at once, before the water-cress
becomes limp.

White Chicken Soup

The water in which a fowl has been boiled
The carcase and bones of the fowl
1 pint milk or cream
1 table-spoon chopped onion
2 table-spoons ” celery
Yolks of two eggs
1 gill chopped and cooked carrot and green peas
Add the bones and carcase of the fowl, the onion, celery and
seasoning to the water in which a fowl has been boiled. Simmer till
reduced to one quart. Strain and thicken with a white roux of butter
and flour. Add the liaison of cream (or milk) and eggs.
Put the cooked carrot and peas in the soup tureen, and pour the
soup over them.

White Veal Soup

2 lbs. knuckle of veal
1 quart water
1 onion
Half pint of milk or cream
2 yolks of eggs
1 table-spoon butter
1 ” flour
Wipe the veal and cut it into small pieces. Cover with cold water
and heat slowly, skimming constantly. Season with salt, three or four
pepper-corns, and a chopped onion. Simmer for three hours until
reduced by half. Strain. Allow it to cool. Remove the fat. Put in the
stew-pan again, and when boiling thicken with white roux made of
table-spoon butter and a level table-spoon of flour (see p. 12). Add a
half pint of milk and eggs. Season again. Serve with fried bread.
 Vegetable Purées
PAGE
Purée of Asparagus 61
” Black Beans 62
” Broad Beans 63
” Carrots 63
” Endive 64
” Green Peas—I 65
” Green Peas—II 65
” Italian Dried Green Peas 66
” Lentils 67
” Onions 68
” Rice 68
” Turnips 69
” Winter Vegetables 69
When passing vegetables or meat through a tammy or fine sieve,
it will be easier if they are kept continually moistened with a little of
the stock or milk with which the purée is to be made.
Purées having been passed through the sieve or tammy, can, if
not required at once, be set aside until wanted. But a purée that has
reached this point must on no account be re-heated or have milk or
cream added to it until just before it is to be served. When re-
heating, if a meat purée, it should not be allowed to boil, or even be
made hotter than is absolutely necessary.
Allow all vegetable purées to boil up quickly for several minutes
after the purée and stock have been mixed. This will clarify them. All
scum should be carefully removed. When this is done, the butter and
milk or cream can be added.
A little white or brown roux well mixed with purées a minute or two
before serving will prevent the actual purée from separating from the
stock.

Purée of Asparagus
1 bundle of asparagus
1 handful spinach
1 small onion
1 quart white stock
¹⁄₂ pint milk or cream
1 oz. butter
Break off all that is tender of each piece of asparagus. Scrape and
wash them. Leave them in cold water for half-an-hour. Drain them.
Put them in a sauce pan with a handful of spinach and a small onion.
When tender take all out and drain again. Add to them a quart of
white stock. Season. Boil gently for ten minutes. Put through a
tammy. Heat slowly again, season and add the butter and scalded
cream. This soup may be deepened in colour by adding a little
spinach colouring (p. 104). Serve with croûtons (p. 103).

Purée of Black Beans

¹⁄₂ pint black beaus

1 quart water
1 carrot (grated)
1 onion
¹⁄₂ head celery
1 table-spoon butter
1 ” brown roux
2 ozs. raw ham or salt pork
2 cloves
 1 bouquet herbs
1 lemon
2 hard-boiled eggs
1 glass sherry
Soak the beans over night. Drain. Put them in a sauce-pan with
one quart cold water, and the ham or pork, the celery, grated carrot,
herbs and cloves pounded. Slice the onion. Fry it in the butter. Add it
to the beans, etc. Simmer for four or five hours. As the water boils
away add cold water to keep it to the same quantity. Put through a
sieve. Return to the fire. Season with salt, pepper, and a little
mustard. Stir in a table-spoonful of brown roux (see p. 13). Just
before serving add the juice of half a lemon and a glass of sherry.
Slice the hard-boiled eggs and half a lemon and put in the soup
tureen. Pour the soup over it. Force-meat balls (p. 104) also may be
served with this purée.

Purée of Broad Beans

1 pint of beans
1 slice of bacon or salt pork
2 sprigs of parsley
3 small onions and one clove
¹⁄₂ head of celery
1 quart water or stock
¹⁄₂ pint milk or cream
1 table-spoon butter
1 ” flour
Boil the beans, one onion, parsley, celery and clove, in one quart
of water or stock, until tender. Rub through a sieve into a basin, and
set aside.
Slice and boil two onions until tender. Drain them. Melt the butter
in a sauce-pan. Add the onions and a little nutmeg and fry until a
good brown, stirring in the flour and mixing it smooth. Add the boiling
milk. Boil for several minutes, stirring all the time. Press through a
sieve, and add to the purée of beans. Season. Heat gently. Serve
with croûtons.

Purée of Carrots
2 large carrots
1 ” onion
1 ” turnip
1 quart beef stock
Scrape the carrots, and slice them finely, using the red outside
part only. Slice the other vegetables. Put all together in a sauce-pan
with the stock. Cook until tender. Rub through a sieve. Return to the
fire. Season and add a small lump of sugar. Serve with croûtons.

Purée of Endive
3 large endives
1¹⁄₂ pints chicken stock
1 table-spoon white roux
¹⁄₂ pint cream or milk
2 ozs. butter
Discard all but the white hearts of the endives. Wash them
thoroughly, and boil them in salt water for ten minutes. Drain them
and put them to stew very gently for quarter of an hour with the
butter, stirring continually. Then add half a pint of white chicken
stock, and simmer for an hour. Pass through a tammy. Return to the
fire and add a pint more stock. Let it boil up. Season. Add the white
roux (p. 12), butter and the boiling cream. Colour with spinach
colouring (see p. 104).

Purée of Green Peas—I

1 pint of green peas
1 turnip
 1 small onion
1 piece of mint
1 oz. of butter
1 quart brown stock
Stew the vegetables with the butter, one pint of stock, and a little
celery seed, until they are quite tender. Rub them through a fine
sieve or tammy. Return to the fire. Add the rest of the stock. Season,
and add a lump of sugar and spinach colouring (see p. 104).
Whenever possible, use half a head of celery finely chopped,
instead of the celery seed.

Purée of Green Peas—II

1 pint of peas
2 small onions
1 cabbage lettuce
1 bouquet herbs
1 quart stock
1 table-spoon white roux
1 gill cream
Stew the peas, onion (sliced), lettuce and a bouquet of herbs in
the butter very gently for ten minutes. Add to them the hot stock.
Bring to the boil. Simmer for half-an-hour. Pass through a tammy.
Re-heat gently. Season. Stir in the roux (p. 12) and a gill of cream.
Serve with croûtons, or add to the soup some young cooked peas.

Purée of Italian Dried Green Peas

¹⁄₂ pint of peas

¹⁄₂ head of celery
1¹⁄₂ pints of stock or water
¹⁄₂ pint cream or milk
1 table-spoon butter
 Soak the peas over night. Put them and the celery, chopped, on to
boil with the water or stock. Boil till very tender. Put through a hair
sieve or tammy. Put back on the fire and heat gently. Season. Colour
with spinach colouring. Just before serving add the butter, in small
pieces, and, when it has melted, the boiling cream. Serve with
croûtons (see p. 103).

Purée of Lentils
1 pint of lentils
1 head of celery
1 onion
1 turnip
1 carrot
1 slice of ham
3 pints stock or water
1 gill of cream
Soak the lentils in water over night. Let the vegetables (which
should be cut up) and the ham stew gently in the butter for ten
minutes. Strain the lentils from the water they have soaked in. Put
them with the ham, vegetables and stock into a sauce-pan. Bring to
the boil and simmer for two hours. Strain off the liquid. Pound and
mash the lentils, etc., and pass them through a sieve. Return them to
the liquid. Boil up again. Add a tea-spoonful of powdered sugar,
seasoning and the cream, scalded. Serve with fried bread (see p.
103).

Purée of Onions
6 onions
1 small turnip
¹⁄₂ head celery
1 quart white stock
2 ozs. butter
 ¹⁄₂ pint cream or milk
12 button onions
Cook the large onions, turnip, celery and butter with the stock until
very tender. At the same time prepare and boil the button onions
until soft. Put the vegetables and stock through a fine sieve. Return
to the fire. Add the cream or milk, scalded, and the button onions.
Season.

Purée of Rice
4 table-spoons rice
1 pint stock
1 pint milk or cream
1 onion
1 carrot (grated)
Bay leaf
¹⁄₂ cup fine bread crumbs
1 oz. butter
Wash and parboil the rice. Add it to the stock with a grated carrot,
the sliced onion (which should have been fried a light brown in the
butter), and the bread crumbs. Simmer for half-an-hour. Pass
through a fine sieve. Return to the fire. Add the scalded milk or
cream, and season. Serve with croûtons (see p. 103).

Purée of Turnip
4 turnips (preferably yellow)
1 large onion
1 carrot
1 piece of celery
4 ozs. butter
1¹⁄₂ pints stock or water
¹⁄₂ pint milk or cream
 Slice the vegetables finely and stew them in the butter. Add half a
pint of the stock hot and simmer until the vegetables are very tender.
Put through a sieve. Add the rest of the stock. Heat. Season. Just
before serving add the scalded milk or cream.

Purée of Winter Vegetables

1 onion
1 carrot
1 large turnip
¹⁄₂ small cabbage
¹⁄₂ head of celery
¹⁄₂ pint of stewed tomatoes
1 quart of water or stock
Bouquet of sweet herb
Table-spoon butter
1 gill cream or milk
Chop all the vegetables but the cabbage and tomatoes very fine.
Put them in a sauce-pan with the water or stock and boil. Cook the
cabbage separately. When the vegetables are tender, add the
cabbage. Simmer ten minutes. Add the tomatoes and a bouquet of
herbs. Boil for quarter of an hour. Rub through a sieve. Return to the
fire. Season. Add the butter and the cream.
 Meat Purées
PAGE
Purée of Fowl à la Reine 73
” ” à la Reine Margot 73
” Hare 74
” Pheasant 75
” Rabbit 75
Whenever a piece of meat or fowl is added to a soup, it must be
added as late as possible, and the soup must not be allowed to boil
after it has been added, or even made very hot. If it boils the purée
will curdle. Should it by accident do so, it is possible to remedy it by
adding a little more stock to the soup, putting it all through a tammy
again, and then warming it gently.

Purée of Fowl à la Reine

1 large tender fowl
¹⁄₄ lb. boiled rice
1¹⁄₂ pints water
¹⁄₂ pint cream
Roast the fowl. Cut off all the meat from it. Chop it and pound it.
Break the bones and carcase of the fowl. Put them and the skin in a
sauce-pan with the water. Bring to a boil and simmer for an hour or
two. Skim and strain. Add it to the pounded meat. Pass through a
tammy. Add the scalded cream. Season.

Purée of Fowl à la Reine Margot