CLINICAL BIOCHEMISTRY 2 MANU2470 Assessment 3: Proposal MAST10005 Calculus 1 BIOL121 ASSIGNMENT 3 SINGLE FAMILY DWELLINGS HI50…

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SUBJECT CODE: BMS302
SUBJECT NAME: CLINICAL BIOCHEMISTRY 2
MODULE 3
Module profile
Name of module:
Endocrinology
Code:
BMS302
Subject relationship:
This is the third of the modules in Clinical
Biochemistry 2.
Prerequisites:
Human Anatomy and Physiology 2 OR
Physiological Sciences, Biochemistry, Clinical
Biochemistry 1
Objectives:
Specific learning outcomes are supplied at the
start of each topic.
Topics covered:
Principles of endocrinology
Disorders of hypothalamic/pituitary axis
Disorders of thyroid gland
Disorders of adrenal cortical gland
Disorders of endocrinology of reproductive
system
Disorders of endocrine control of calcium,
phosphate and magnesium metabolism
Resources provided:
Study guide, prescribed textbook and readings.
Learning strategies:
This module is based on material from the
prescribed text and other texts. The study
guide provides expected student learning
outcomes for each topic, which provide
direction in your reading.
Assessment:
MSE & ESE
Practicals/exercises/case studies
Module author:
Associate Professor Dr Geoff McKenzie and
Dr Phillip Bwititi
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Introduction
Higher animals maintain a relatively constant internal environment despite large fluctuations
in external surroundings and such constancy or homeostasis is maintained mainly by neural
and endocrine systems.
This module commences by considering the endocrine system in general including:
 the range of hormones, chemical structures and mode of action; and
 the role of the hypothalamus and anterior pituitary gland in controlling the peripheral
endocrine glands.
Subjects you have studied provide background knowledge that is extended in this
module. Before you start you may need to review the following topics:
Human Anatomy and Physiology or Physiological Sciences:
 Location and structures of endocrine organs
 Function of hormones
 How hormones exert activity.
From Biochemistry:
 Structures of amino acids, peptides/protein and steroid hormones
 Hormones as regulators of metabolism.
Dynamic (stimulation/suppression tests also challenge tests): In this module you will learn
about dynamic tests. We are interested in why such tests are done i.e. rationale or reasons for
doing the tests. The ‘recipe’ i.e. knowing the exact dosage (these are sometimes given in text)
is not necessary as this can change from place to place and dosages are governed by age and
weight, among others. Know how the test is done i.e. is it oral or i.v. and have some idea of
the duration of the test i.e. if the test is carried out e.g. over-night or hourly.
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Topic 1 General principles of endocrine function
This topic is a re-cap of endocrine physiology and anatomy and biochemistry and forms the
basis of understanding clinical (rationale/reasons for measurement) endocrinology and
analytical (measurement) endocrinology. Refer to previous texts and notes.
Assumed previous knowledge
Biochemistry
 Structures and functions of hormones
Human Anatomy and Physiology/Physiological Sciences
 Location and structure of endocrine organs
 How endocrine organs work
Learning outcomes
 describe basic concepts of endocrinology;
 describe major structural groups of hormones and identify hormones that fall into
each group;
 describe mode and control of synthesis, storage, release and transport of hormones;
 describe receptors and their significance in hormone action and how hormones
interact with receptors to effect a biochemical response;
 identify hormones with plasma membrane and intracellular receptors;
 analyse the hypothalamus-anterior pituitary gland-peripheral gland axis in control of
hormone secretion;
 analyse the basis or rationale for investigations of endocrine diseases i.e. evaluate
endocrine investigations.
Introduction and classification of hormones
This section is mostly catch-up from physiology and biochemistry
The neural system transmits electrical signals in the form of action potentials through neurons
that release neurotransmitters in synapses and in neuromuscular junctions. This mode of
communication is fast and precise. The endocrine system releases hormones into the blood,
which travel and effect activities through specific receptors in target organs. The activated
receptor triggers e.g. changes in the activities of enzymes in a cell (signalling pathway),
resulting in a biochemical response. Conversion of neural or hormone-mediated message into
an activated signalling pathway in a receptor-bearing cell that ultimately results in a
biochemical response is called signal transduction.
Endocrinology deals with Biochemistry/Physiology and Clinical Endocrinology deals with
pathophysiology of endocrine systems and the measurements of hormonal responses. Topics
in Endocrinology and Clinical Endocrinology cover synthesis, storage, release, transport and
response of target cells to hormones.
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Measurement of hormones is covered in Module 1.
Classification of hormones
You are familiar with hormone structures and examples of hormones belonging to the various
groupings (from Biochemistry and Physiology subjects).
a. Peptide or protein e.g. insulin: These vary in size and some are small, for example,
ADH has 9 amino acids while human proinsulin has over 80 amino acids. These
hormones are in most cases synthesised in the pre-form and since they are proteins,
they are water-soluble and because they are large and charged, they do not penetrate
membranes. Therefore, their receptors are normally on plasma membranes. Another
subclass of protein hormones are glycoproteins, which are a result of post-synthesis
glycosylation; in this class are hCG, TSH, FSH and LH. Synthesis of peptide
hormones normally involves one cell type and one organ.
b. Steroids, e.g. cortisol: Steroid hormones are small and lipid-soluble and circulate in
plasma bound to proteins e.g. cortisol is transported by cortisol binding globulin.
They can penetrate membranes, hence their receptors are normally cytosolic and
nuclear. Steroid hormones share a common pathway of synthesis from cholesterol and
are mainly synthesised in adrenals and gonads. Since steroid hormones are lipidsoluble they have easy access to CNS. Oestrogens and androgens affect neural
development in young and behaviour in adults.
Complete biosynthesis of steroids can involve more than one cell or organ, e.g.
synthesis of Vitamin D3 occurs in the skin, then liver and ultimately in the
kidney. Synthesis can also be carried out by more than one organ, e.g. androgens are
synthesised by adrenals and gonads while progesterone is synthesised in the ovary and
placenta. The active form of steroid hormones may be represented by more than one
chemical form and this is common in androgens and oestrogens. Agonists and
antagonists for steroid hormones are wide-spread in nature, hence can be abused or
used in therapy.
c. Amines, e.g. thyroxines (T4, T3) and catecholamines (adrenaline, noradrenaline and
dopamine): Have characteristics in-between steroid and peptide hormones and are
synthesised from tyrosine. Thyroxines have nuclear receptors while catecholamines
have plasma membrane receptors.
What do hormones do?
One function – multiple hormones
In the control of plasma glucose level, several processes are involved: Carbohydrate intake,
glycogenolysis, gluconeogenesis, glycogenesis and glycolysis (you should be able to define
these terms). These processes are controlled by insulin, glucagon, catecholamines, cortisol
and thyroid hormones.
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One hormone – multiple functions
Parathyroid hormone controls Ca2+, HPO42- and Mg2+ metabolism in bone, GIT and kidney.
Insulin controls carbohydrate, protein, lipid and Na+ metabolism.
What governs hormonal secretions?
Basal
Even when no trophic or homeostatic challenge exists, some hormones are continuously
produced and neuro-regulation is an important factor in basal secretions.
Stimulation
a. When the level of a circulating substrate changes, a hormone is released.
i. Low plasma Ca2+ level increases PTH release.
ii. High plasma glucose concentration increases insulin release.
b. Increased level of trophic factor or hormone
i. Hypothalamus —TRH → Anterior pituitary — TSH → Thyroid — Thyroid hormones →
Target cell
One releasing factor or hormone gives rise to one trophin, that gives rise to one hormone
ii. Hypothalamus — CRF → Anterior pituitary — ACTH (Corticotrophin) → Adrenal —
Cortisol and androgens → Target cell
One releasing factor or hormone gives rise to one trophin, that gives rise to two hormones
iii. Hypothalamus — GnRH → Anterior pituitary — LSH and LH → Ovary — Oestrogen
and progesterone → Target cell
One releasing factor or hormone gives rise to two trophins, that gives rise to two hormones
It is important to understand these concepts and if you consider point (b ii) it means that a
disease that changes the ACTH level (can be a tumour) will result in alterations in cortisol
and androgen levels. Remember that cortisol controls glucose level and androgens influence
development of sexual characteristics.
Feedback
Endocrine cells need to know when to secrete and when to stop secreting a hormone.
Endocrine systems are therefore governed by feedback, which is a fundamental property of
biochemical or physiological processes. Changes in the level of a circulating metabolite or
hormone can stimulate or inhibit the release of a hormone that controls it. Such mechanisms
allow resistance in deviations from pre-set ranges in biological systems (thermostat).
Feedback can be negative or positive and occurs at different levels as shown:
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a. Substrate: High plasma glucose level stimulates insulin secretion (positive feedback),
while increased plasma calcium level inhibits parathyroid hormone secretion
(negative feedback). Progesterone also exerts positive feedback on FSH; this in part
triggers ovulation.
b. Hormonal: Hormone controlling another hormone, e.g. TSH controls of thyroxine
synthesis and secretions.
Feedback is crucial in endocrinology and will constantly be referred to.
Synthesis/Storage/Release/Transport/Receptors
Synthesis
Polypeptide hormones are synthesised as part of large proteins, which are cleaved to produce
the active molecule. The precursor is known as a pro-hormone and in some cases the initial
protein is synthesised as an even larger protein known as a pre-pro-hormone.
Steroid hormones are synthesised from cholesterol, which is synthesised from acetate.
Amide hormones (thyroxines) and catecholamines are synthesised from tyrosine.
Storage
Protein and polypeptide hormones are normally stored as secretory granules in the endocrine
cell cytoplasm. Steroid hormones are probably not stored, while in other groups there is
variable storage.
Release
Protein and polypeptide hormones are released by exocytosis, while steroids diffuse out of
the endocrine cell into extracellular fluid. Hormones have different half-lives and such
information is important because it governs analysis, e.g. a hormone with a short half-life
would be difficult to analyse. What is also important are the patterns of synthesis and
secretion of hormones. Cortisol is secreted rhythmically and in a pulsatile manner and the
level of plasma cortisol at mid-night is about a quarter of the level around 8am. We also need
to consider factors such as the effects of menstrual cycle on female sex hormone levels, as
well as environmental factors and stage of growth. Knowledge of these factors is important in
therapy, analysis and in interpretation of endocrine data.
Transport
Polypeptide and protein hormones are water soluble and are transported as pre-pro-hormones,
pro-hormones, or as active molecules. Steroid and thyroid hormones are bound to plasma
proteins and a dynamic equilibrium exists in the blood between bound and free fractions of
the hormone. There are specific proteins that bind hormones e.g. thyroid binding globulin
(TBG) for T4 and corticosteroid binding globulin (CBG) for cortisol. The percentage of the
fraction that is bound varies between proteins e.g. 60% of aldosterone is protein bound and
the figure for T4 is 99%. Binding of hormones to proteins has pathophysiological importance
in ‘pseudo cases’, in which the total hormone level changes simply because the concentration
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of protein has changed. Note: it is the free fraction that exerts physiological/biochemical
activity. One protein can bind more than one hormone and one hormone can be bound by
more than one protein. Transcortin or CBG has high affinity for progesterone and cortisol.
Sex hormone binding globulin (SHBG) binds testosterone, oestradiol and
dihydroxytestosterone.
Receptors
Receptors on cell surfaces respond to factors that are chemical and physical. The distribution
of receptors varies depending on the function of the hormone. Glucocorticoid receptors
are found in a wide variety of cell types, consistent with their function. A receptor has a
binding or recognition site and the binding of ligand to receptor triggers intracellular events.
Receptors are for recognition and activation as well as for regulation of hormonal action.
Hormones, neurotransmitters and local mediators act through receptors and a molecule that
binds to a receptor is a ligand. In Endocrinology, some receptors are enzymes that e.g.
phosphorylate protein substrates inside the cell while others are linked to G-proteins.
Because of their small size and hydrophobicity, steroid and thyroid hormones normally
diffuse directly into cells and bind to intracellular receptors in the cytosol or nucleus. The
activated hormone-receptor complex then binds to DNA, regulating transcription by
induction or repression. Proteins bind to cell surface receptors triggering production of
second messengers that alter transcription, translation and post-translation events. In receptormediated hormonal action, the hormone-receptor complex does not leave the membrane. It
activates a second messenger and the hormone-receptor complex is eventually endocytosed
into the cell after exerting activity.
From the above it should be clear that endocrine diseases arise because of:
a. Abnormal synthesis of a hormone
b. Inappropriate release of a hormone
c. Inappropriate transport of a hormone
d. Receptor defects: numbers or structures
e. Inappropriate intracellular signalling
f. Ectopic release of a hormone (secretion of a hormone from a site where it is not normally
secreted from; this occurs in tumours).
Note: some substances that are not hormones can have hormonal-like activity. Some
antibodies can bind to hormones in circulation or to receptors and this can stimulate or inhibit
hormone-mediated processes.
Understanding these concepts forms the basis of diagnosis and therapy in endocrinology.
Consider hypothalamic (cranial) and nephrogenic diabetes insipidus, as well as Type 1 and 2
diabetes mellitus. Interest in endocrinology is also growing in the field of sports medicine
because hormones and/or their analogues can be substances of misuse and abuse.
Signal transduction
Activation of a receptor triggers a cascade of signalling within the cell and this is achieved by
2nd messengers, which are products of intracellular enzymatic actions. Regulatory proteins
(G-proteins) that bind GTP are used in many cells to couple cell surface receptor activation to
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generation of 2nd messengers. The inability to generate 2nd messengers is implicated in a
number of diseases. For instance, in pseudohypothyroidism Type 1a, administration high
level of PTH fails to elevate cAMP because these patients have low level of G protein
subunits.
Several receptor types can exist for a hormone and several hormones can share a receptor but
with different affinities – catecholamines are examples of this. Although steroids have
specific receptors, they bind to receptors of other steroids (but with less specificity) and this
is called spill-over. Glucocorticoids and mineralocorticoids bind to each other’s receptors.
Increased cortisol; a glucocorticoid will mimic mineralocorticoid excess by spill-over
manifesting as Na+ retention, K+ loss and hypertension.
Just like the concentrations of hormones vary, so do concentration and affinity of receptors
and this results in down-regulation and up-regulation. This also regulates the effectiveness of
a given concentration of a hormone by shifting the hormone-receptor curve to the left or
right. For instance, if insulin concentration increases, the body might respond by reducing the
number of insulin receptors; this controls insulin effectiveness.
Autoantibodies to receptors are common e.g. autoantibodies to insulin receptors have been
found in some patients with insulin resistance. These antibodies bind to a different site on the
receptor, inhibiting insulin binding. Some antibodies also bind to receptors and mimic the
metabolic effects of the hormone and over a period they desensitise intracellular signalling. In
Grave’s disease, antibodies bind to TSH receptors on the thyroid, continuously stimulating
the thyroid gland.
Some of the ways by which hormones act on target cells:
1. Affecting protein biosynthesis, acting as inducers or repressors. Steroid hormones
and thyroid hormones act in this manner.
2. Affecting the activity of enzymes, without affecting enzyme synthesis. Most
peptide hormones plus adrenaline belong to this group.
3. Affecting the permeability of cell membranes. An example of this category is the
way in which vasopressin (ADH) increases the permeability to water of parts of the nephron.
Control of the endocrine system
As mentioned, feedback systems are crucial in maintaining homeostasis and the
hypothalamic-pituitary gland-target glands axes demonstrate long and short feedback loops.
Negative and positive feedbacks can also be demonstrated by examining the actions of sex
steroids. It is important that the level of a hormone in the blood is appropriate to the
physiological status of the body. In addition, hormone secretion must be capable of being
turned on/off quickly in response to physiological changes. An example of such control is
seen in hormones that control blood glucose level. After a meal, blood glucose level rises,
stimulating the secretion of insulin to remove glucose from the blood, while during fasting,
adrenaline and glucagon are secreted in response to low blood glucose level. These two
hormones promote the input of glucose into the bloodstream. The net effect on blood glucose
level is a balance between the actions of two sets of opposing hormones.
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Few endocrine glands have such a direct control system via the blood and therefore other
control systems have evolved. These involve neural control of hormone secretion via the
hypothalamus and the anterior pituitary and the system works as follows:
 the hypothalamus secretes hormones (releasing factors) which act on the anterior
pituitary;
 the anterior pituitary gland is stimulated to secrete a trophic hormone, which is
released into the bloodstream;
 the peripheral endocrine gland is stimulated to secrete hormones, which exert the
appropriate metabolic or physiological effect; and
 high levels of these hormones in the blood feedback on the hypothalamus and
pituitary to inhibit the secretion of the releasing factor and trophic hormones,
respectively.
The way this system operates is illustrated by the example of thyroid hormones shown below
(see also Figure 8.3 of prescribed text). Further discussion of the action of the hypothalamus
and pituitary gland is given in the next section.
Figure 3.1 Hypothalamic-anterior pituitary gland thyroid gland axis (colostate.edu)
(+) is stimulation; (-) is inhibition
Laboratory investigation of endocrine function
Clinical Biochemistry investigations of endocrine disorders involve:
1. Measurement of blood levels of hormones emanating from the peripheral gland.
Measuring blood hormone level only tells us that the amount of hormone secreted is
inappropriate, but does not pinpoint the cause of the malfunction; it may be a lesion
on the gland, the pituitary or the hypothalamus or it may be loss of control of
secretion. To distinguish these possibilities other laboratory measurements are
necessary.
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2. Measuring the urinary output of metabolites of a hormone.
These methods are not used a lot these days.
3. Measurement of blood levels of trophic hormones.
Such measurements allow distinction of disorders emanating from the pituitary from
those in the peripheral gland. For example, in hypothyroidism, low blood TSH levels
suggest a lesion in the anterior pituitary, whilst normal/high blood TSH levels that the
disorder is located in the thyroid gland.
4. Dynamic tests of endocrine function.
Suppression or stimulation tests assess hormonal control systems. A good
understanding of the normal control mechanism (hypothalamus-pituitary-gland axis)
and the specific effect of the stimulator or suppressor is critical for a successful
outcome.
5. Measurement of antibodies.
A number of endocrine diseases are associated with autoimmunity against endocrine
organs, e.g. some forms of Type 1 diabetes mellitus hence antibodies can be
measured.
6. Receptors
Reduction or increase in the number of receptors is associated with changes in
sensitivity to hormones; hence receptor density can be measured.
7. 2nd messengers
Some diseases occur in the presence of normal and in most cases elevated hormone
level, but the internal messenger is not activated hence internal messengers such as
cAMP are measured.
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Topic 2 Hypothalamus and pituitary gland hormones dysfunction
Rae P., Crane M., & Pattenden R. (2018). Clinical Biochemistry: Lecture
notes (10th ed.), Chapters 4 & 7. Wiley-Blackwell.
Hypothalamic-pituitary gland axes (aneskey.com)
See also Figure 7.1 in prescribed text
Learning outcomes
 describe structure and regulation of the hypothalamic-pituitary gland axis, including
the distinction between adenohypophysis (anterior lobe) and neurohypophysis
(posterior lobe);
 describe structures and functions of major hormones secreted by hypothalamus that
act on anterior pituitary gland and their clinical significance;
 describe structures and functions of hormones secreted from posterior and anterior
pituitary glands and their clinical significance;
 describe and evaluate assessment of anterior pituitary gland function;
 analyse diabetes insipidus (DI) and distinguish between hypothalamic and
nephrogenic DI;
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 evaluate renal tubular function in terms of water and osmolality regulation in regards
to fluid deprivation and vasopressin tests.
Pituitary hormones
The hypothalamus is part of the brain but unlike other parts of the brain, the hypothalamus is
not sealed off from the rest of the body. Metabolites in the blood reach the hypothalamus
hence the hypothalamus can monitor osmolality, temperature, etc. A number of diseases
present as pain, fever and weight changes. These parameters are also controlled by the
hypothalamus.
The pituitary gland is made up of two large lobes (anterior and posterior) with a smaller
compartment; the intermediate lobe between the two. A stalk of nerve fibres and blood
vessels (infundibulum) connect the hypothalamus and pituitary gland. The hypothalamic
control of pituitary function differs between the anterior and posterior lobes.
Anterior pituitary gland and associated infundibulum is called adenohypophysis while
posterior pituitary gland and associated infundibulum is called neurohypophysis.
The anterior lobe is controlled by releasing factors (releasing hormones) and erior
lobe, being a direct neural outgrowth of the hypothalamus, is controlled by nerve activity
directly. The release of hormones from the anterior pituitary gland is controlled by
hypothalamic hormones (factors). These releasing and inhibiting hormones are transported
from the hypothalamus to the anterior pituitary through the hypothalamo-pituitary portal
system. This allows relatively small amounts of releasing hormones to be preferentially
sequestered to the target gland (anterior pituitary) without being diluted in the systemic
circulation.
Posterior pituitary gland is an outgrowth of hypothalamus and its neural tissue. Axons from
hypothalamus pass through infundibulum into posterior pituitary gland. Posterior pituitary
gland hormones are synthesised in hypothalamus and are only released from posterior
pituitary gland.
erior lobe releases two hormones: ADH or vasopressin and oxytocin, under direct
neural stimuli from the hypothalamus. Hypothalamic ADH (vasopressin) was covered in
BMS207: Clinical Biochemistry 1 under Osmoregulation, please re-cap. Oxytocin is
covered here and also in topics dealing with reproduction.
Posterior pituitary gland
Responsible for:
a. Antidiuretic (water conserving).
b. Vasopressic (raising blood pressure) through vasoconstriction.
c. Oxytocic (uterus contracting).
These characteristics are exhibited by vasopressin (antidiuretic hormone) and oxytocin.
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Vasopressin and oxytocin are nanopeptides with cys-cys at 1-6 positions. There is crossbiological reactivity between vasopressin and oxytocin. Vasopressin is found in all mammals
and we have arginine at position 8 hence arginine vasopressin (AVP) but in pigs arginine is
substituted by lysine giving lysine vasopressin (LVP). Other neurohypophyseal peptides are
also found in various species e.g. valitocin and aspartocin in dogfish.
Figure: amino acids sequence of oxytocin and ADH
As mentioned, ADH and oxytocin are nanopeptides with cys-cys at 1-6 position. Because of
similar structures, there is cross-reactivity between ADH and oxytocin (understand and do
not memorise structures). ADH (vasopressin) is found in all animals and in most of these
animals arginine is at position 8 hence arginine vasopressin (AVP). Arginine is substituted by
lysine in pigs to give lysine vasopressin (LVP). In Pathology, vasopressin comes as AVP or
LVP (please know the differences especially when you carry out immunoassays). Other
neurohypophyseal peptides (similar to ADH and oxytocin) are found various animal species.
In birds, these peptides are involved in egg laying just as oxytocin is involved in parturition
in humans. Should you work or if you work in Animal Pathology you will come across these.
Synthesis and release
Vasopressin and oxytocin are synthesised as prohormones in the cell bodies of magnocellular
neurones, which are concentrated in 2 structures: supraoptic and paraventricular nuclei. The
prohormones are then transported bound to neurophysin in vesicles through the axons to neural
lobes in posterior pituitary gland for release. In the neural lobe, oxytocin and vasopressin are
stored as granules. Nerve stimulation in the hypothalamus propagates along the axon and
triggers hormone release. The nanopeptides and neurophysins are thought to be released in
equi-molar amounts.
The neurohypophyseal neurones are controlled by cholinergic and noradrenergic
neurotransmitters and some neuropeptides. Acetylcholine stimulates ADH and oxytocin
release. Hence, the antidiuretic effect of smoking is due to nicotinic acid receptor stimulation.
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β adrenergic stimulation are inhibitory to oxytocin release. Hence, stress inhibits milk-let down
reflex in humans and other animals.
Regulation of release
Vasopressin (ADH)
AVP acts on various organs in different animals increasing water retention. For instance, in
some amphibians AVP acts on the skin and bladder.
a. Osmolality
One of the cardinal functions of the hypothalamus is maintenance of ECF osmolality
within +2%. Mean plasma osmolality is 282 mosmol/Kg in humans and the osmotic
threshold for AVP secretion is 287 mosmol/Kg. Water loading inhibits AVP release.
These effects are mediated by the hypothalamic osmoreceptors.
b. IVV
Reduction in IVV of greater than 10% of the normal stimulates AVP release. Severe
reduction in IVV will override osmotic response.
Stretch receptors are located in the left atrium of the heart and are sensitive to distension
of the atrium caused by filling of the heart with blood from the pulmonary circulation.
These respond to moderate reduction in IVV. On the other hand, baroreceptors located
in the carotid sinus, are sensitive to blood pressure in the carotid arteries. These appear
to respond to large decrease (sufficient to cause hypotension) in IVV.
Blood osmolality and volume are integrated in stimulating AVP release. Water loading
increases IVV and decreases blood osmolality resulting in lowering of AVP. Reduction
in IVV through haemorrhage stimulates AVP release resulting in a decrease in
osmolality. In severe blood loss, maintenance of IVV is preferred to maintenance of
osmolality. Baroreceptors will therefore override osmoreceptors.
Other factors affecting ADH release:
Stimulators: Exercise, emotion, stress, pain and fright, nicotine, hypoxia, haemorrhage, heart
failure, liver cirrhosis.
Inhibitors: Alcohol, caffeine.
Diseases associated with vasopressin (ADH)
Diabetes insipidus (DI)
DI is associated with diuresis, urine excretion can reach as much as 25 L/day. The urine is of
very low osmolality. The patients become dehydrated and hypotensive.
DI is classified into:
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Hypothalamic or cranial DI: due to decreased vasopressin synthesis and release by the
hypothalamus or decreased release by erior pituitary gland. Plasma level of vasopressin
is therefore low. Injection of vasopressin in these patients is followed by a response.
Nephrogenic DI: due to the inability of the renal tubule to respond to vasopressin. Because of
feedback mechanism, the plasma vasopressin level is high. Administration of vasopressin in
these patients has no effect.
Assessment of tubular dysfunction in terms of water and osmolality: fluid deprivation
and vasopressin tests
There are various renal tubular dysfunction disorders and some result in the inability if the
renal tubules to concentrate urine i.e. conserve water (Table 4.3 of prescribed text). This
therefore calls for testing the urine concentrating ability of the kidney.
Urine osmolality and/or specific gravity and urine volume are measured coupled with
measurement of blood osmolality, electrolytes and other renal function tests. Pages 48-50 of
prescribed text detail assessment of renal tubular function in relation to urine concentrating
ability.
As mentioned, mean plasma osmolality is 282 mosmol/Kg (275-295) and urine osmolality
varies (50-1 250 mosmol/Kg). This variation is determined by the need to maintain blood
volume and osmolality and electrolyte levels.
In these patients first line investigations include measurement of early morning urine
osmolality. Normally, early morning urine osmolality should be >800. Further testing calls for
controlled studies: fluid deprivation and/or vasopressin tests.
Water/fluid deprivation test
Patients refrain from water intake over-night (normally starting after 10pm). Collect urine and
blood specimens at intervals until 3pm the following day starting the morning (~ 8am). Patient
is also weighed at intervals.
Protocol varies from place to place and patient needs constant monitoring.
Interpretation: No increase in plasma osmolality (275-295) over the water deprivation period.
Urine osmolality elevates > 800 and urine volume progressively goes down.
Patients with DI fail to concentrate urine i.e. fail to elevate urine osmolality and fail to reduce
urine volume.
Vasopressin/ADH test
Useful in distinguishing nephrogenic DI from Cranial/hypothalamic DI.
1-deamino, 8-D-arginine vasopressin (DDAVP), is a synthetic analogue of vasopressin.
Again, protocol varies from place to place and patient needs constant monitoring.
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After fluid deprivation the patient is given water (volume depends on age and how they
responded to fluid deprivation). DDAVP is injected after the water deprivation period and urine
is collected hourly for 3 hours.
Interpretation: Patients with Cranial/hypothalamic DI do not respond to water deprivation but
respond to DDAVP administration by increasing urine osmolality and reducing urine volume
after DDAVP administration.
Patients with nephrogenic DI fail to concentrate urine during fluid deprivation and also do not
respond to DDAVP i.e. fail to concentrate urine (urine osmolality remains low and urine
volume high).
Note: Patients with psychogenic DI should respond to water deprivation and the DDAVP test
but the response may be moderate.
Oxytocin
Also covered in reproductive endocrinology. Please note that there will not be stand-alone
exam question on oxytocin. Disorders of oxytocin secretion are rare and not clinically
important for purpose of this subject. You will come across oxytocin in research and in
animal farming.
Actions of oxytocin
Milk let-down (milk injection) reflex: Milk appears at the nipple after a suckling stimulus that
initiates neurogenic reflex which is transmitted from the nipple to hypothalamus where
oxytocin release is triggered.
Oxytocin causes contraction of myoepithelial cells that encircle mammary acini expelling
milk into nipple. Pain, stress or fright inhibits milk let-down.
Parturition: Uterus-contracting activity of oxytocin was one of its functions to be first
identified. This property is also been utilised to induce labour. Oxytocin reduces bleeding
during/after delivery. In birds, reptiles and amphibians, arginine vasotocin is a potent
stimulator of oviduct or uterus, hence is involved in egg laying.
Mating: Oxytocin stimulates rhythmical contractions of the vagina and uterus to aid sperm
motility hence fertilization.
Maternal behavior: In the mother, oxytocin is thought to reduce anxiety of exposure to the
cry of newborn. Oxytocin also appears to act on the brain centers that control maternal
behavior stimulating maternal instincts and behavior.
Other hormones secreted by the hypothalamus (not examinable)
The hypothalamus secretes other peptides/compounds whose physiologic functions are not
clear. These peptides/compounds are also found in other parts of the brain as
neurotransmitters. These include substance P, neurotensin, angiotensin 11, leu-enkephalin,
met-enkephalin, , and  endorphins, VIP, bombesin etc.
17
The hypothalamus is responsible for satiety and hunger. Lesions of the hypothalamus is
associated with phagic changes, weight changes and at times obesity. Hypothalamic
hormones and peptides associated with food intake include orexins, neuropeptide Y, leptin,
peptide YY, galinin and noradrenaline. Hypothalamic peptides that suppress food intake are
CCK, glucagon like peptide-1, CRH, insulin, bombesin, serotonin and urocortin.
Anterior pituitary gland
Neurotransmitters are released in the hypothalamus from neurones arising from the brain.
These neurotransmitters stimulate synthesis and release of hypothalamic peptides or factors
(if structure is not known) that are transported to anterior pituitary gland were they stimulate
synthesis and release of anterior pituitary hormones.
The anterior pituitary gland is connected to the hypothalamus by hypothalamic-pituitary
portal system. This allows trophic factors or peptides from hypothalamus to reach anterior
pituitary gland without being diluted in systemic circulation. This also ensures that these
factors do not target other organs in the body.
Hypothalamic hormones have low affinity for pituitary gland receptors. Low affinity ensures
that the hormones do not bind tightly. If a chemical binds tightly to a receptor it means that
the biochemical activity will persist and you do not want that to occur. Hypothalamic
hormones have short half-lives. This is also important because again you do not want the
activity to persist. However, short half-lives cause problems in analysis because such
hormones are unstable and difficult to measure.
The hypothalamic hormones acting on anterior pituitary gland have stimulatory or inhibitory
effects on cell differentiation of pituitary gland and on synthesis of hormones in pituitary
gland. The hormones include corticotrophin releasing hormone, thyrotrophin releasing
hormone, luteinizing hormone releasing hormone (gonadotrophin releasing hormone) and
growth hormone releasing hormone, which are stimulatory. Dopamine is a non-peptide
secreted by the hypothalamus to affect the anterior pituitary and it inhibits prolactin secretion.
Stimulatory and inhibitory hormones interact to modulate hormone synthesis and release in
the anterior pituitary gland. This is seen in the synthesis and release of PRL, TSH and GH.
Releasing hormones can also stimulate release of more than one pituitary gland hormone:
• TRH stimulates PRL, ACTH, and GH.
• LHRH releases both LH and FSH
• Somatostatin inhibits secretion of GH and TSH.
• Dopamine inhibits PRL, TSH, GH and gonadotrophin secretion.
Feedback loops of hypothalamic-anterior pituitary gland–endocrine organ axis
• Development and metabolism
• TRHThyrotrophin (TSH)Thyroxines
• Stress
• CRHACTHCortisol
• Growth
• GHRHGH, IGF-1Somatostatin
• Reproduction
18
• GnRHLH, FSHOestradiol/testosterone
Prolactin (also covered in reproductive endocrinology): a polypeptide that is produced by
lactotrophs and is structurally similar to GH and placental lactogen. In women its’ major
function is initiation and maintenance of lactation and suppression of fertility during
lactation.
It appears there is no hypothalamic stimulator of prolactin but dopamine from the
hypothalamus inhibits prolactin secretion
In all sexes, prolactin controls gonadal function. Its’ secretion is suppressed by prolactininhibitory factor (PIF), which has dopamine-like activity. TRH increases prolactin secretion.
Highest levels are during sleep and lowest levels in the morning. Prolactin is increased in
pregnancy and in stress.
Hyper-prolactinaemia causes menstrual irregularities, infertility, low libido and hirsutism and
acne, vaginal dryness thus painful sex in females while in males there is enlargement of
breasts and lactation and infertility.
In males, low prolactin levels are associated with low libido, erectile dysfunction and low
sperm count while in females there is ovarian dysfunction, lactation failure after birth.
Macro-prolactin: There are patients with auto-antibodies (usually IgG) to prolactin and these
antibodies combine with prolactin in the body to form a fairly stable prolactin-IgG, which has
a long half-life. This therefore elevates total prolactin levels in blood, which can wrongly be
taken as true hyper-prolactin.
Growth hormone: a polypeptide that is secreted by somatotrophs and its’ release is positively
controlled by ghrelin and GHRH from hypothalamus. Its’ release is also stimulated by ghrelin
that is produced fundus of stomach and is one the hormones that control feeding.
GH release is suppressed by high dose of glucose and stimulated by hypoglycaemia. This
property is used in dynamic tests for acromegaly and gigantism as well as dwarfism (read
from page 94 on GTT in prescribed text). Insulin hypoglycaemia test or insulin tolerance test
is used to stimulate GH in investigation of GH deficiency.
Insulin-like growth factor (IGF-1) and GH act on hypothalamus and anterior pituitary gland
via negative feedback to control GH synthesis and release.
GH release is elevated during sleep, low levels during the day make measurement difficult.
Growth hormone acts to:
increase: protein synthesis, lipolysis, growth, glucose synthesis in the liver
reduce: glucose uptake.
Actions of GH are mainly stimulation of hepatic IGF-1. IGF-1 is similar to insulin
structurally and stimulates growth in almost all cells.
GH & IGF-1 are useful in assessment of GH excess & deficiency
19
Dynamic tests for GH investigations
GH release is suppressed by high dose of glucose and stimulated by hypoglycaemia. This
property is used in dynamic tests for acromegaly and gigantism as well as dwarfism (read
from page 94-96 on GTT in prescribed text).
GTT is used test to suppress GH release in investigation of GH excess (pages 95-96 of
prescribed text).
Insulin hypoglycaemia test/insulin tolerance/GH provocation tests is used to stimulate GH in
investigation of GH deficiency (page 103, 289-290 of prescribed text). Other commonly used
provocative tests (pages 289-290 of prescribed text) are arginine whose stimulation effects of
GH secretion are possibly mediated by arginine’s suppression of somatostatin (growth
hormone inhibiting hormone) secretion. Somatostatin is normally released from
hypothalamus and inhibits GH release. Another provocative test uses clonidine (alpha
adrenergic agonist), which stimulates the release of GHRH, from hypothalamus that then acts
on anterior pituitary gland to stimulate release of GH.
Please note that in the investigation of the hypothalamic-anterior pituitary gland axis e.g. if
hypopituitarism is suspected several hormones in this axis are assessed. The
products/hormones of the anterior pituitary gland are also measured. The hormones from the
anterior pituitary gland such as GH and prolactin have direct effects on tissues, this is also
assessed.
Lastly, the anterior pituitary gland hormones have stimulatory effects on other endocrine
glands and thus the hormones from these glands (adrenocortical, thyroid and testes) are
measured and well as the effects of these hormones on tissues/metabolism.
As mentioned, the anterior pituitary gland produces hormones that affect several endocrine
glands and these will be covered i.e. hypothalamic-anterior pituitary gland-endocrine gland
axis. Anterior pituitary gland hormones include:
ACTH, FSH, LH and TSH
beta-lipotropin, endorphins
Please note that ACTH, lipotropin and endorphins are produced from a common precursor
i.e. from cleavage of pro-opiomelanocortin (POMC).
Because the hypothalamus produces hormones/factors that affect the anterior pituitary gland,
which in turn produces hormones/factors that affect various endocrine organs, most of the
topics in this module will refer to the hypothalamus/anterior pituitary axis. A disease in this
axis therefore affects endocrine glands such as thyroid, adrenal and gonads. More on
the anterior pituitary gland will be covered in several topics in this module:
• Hypothalamic-anterior pituitary gland-thyroid gland axis
• Hypothalamic-anterior pituitary gland-adrenocortical gland axis
• Hypothalamic-anterior pituitary gland-ovarian gland axis
• Hypothalamic-anterior pituitary gland-testicular gland axis
20
21
Topic 3 Thyroid gland hormones dysfunction
Rae P., Crane M., & Pattenden R. (2018). Clinical Biochemistry: Lecture
notes (10th ed.), Chapter 8. Wiley-Blackwell.
Assumed previous knowledge
Biochemistry
 Functions of hormones
 Oxidative phosphorylation
Human Anatomy and Physiology / Physiological Sciences
 How endocrine organs work
 Location and structure of the thyroid gland
Learning outcomes
 outline the chemical structures of thyroxine (T4), triiodothyronine (T3) and reverse
triiodothyronine (rT3);
 describe synthesis and transport of thyroid hormones;
 analyse control of thyroid hormone secretion, in terms of hypothalamus-anterior
pituitary-thyroid gland axis;
 describe structure, regulation and clinical significance of TSH and TRH
 describe actions of thyroid hormones;
 analyse the pathological conditions affecting thyroid function, including;
(a) range of disorders resulting in hypothyroidism
(b) range of disorders resulting in hyperthyroidism
(c) describe goitre and its causes
 evaluate tests to diagnose and monitor thyroid function, including;
(a) role of total and free T4 and T3 tests
(b) physiological and pathological factors affecting levels serum binding proteins
(c) role of serum TSH (thyrotropin) tests
22
Importance of thyroid hormones
Understanding the physiology and biochemistry of thyroid hormones is important because:
a. Symptoms of the disorders of thyroid hormones are varied due to the fact that thyroid
hormones affect almost every organ and the effects are varied.
b. Disorders of thyroid hormones are common and can be managed by appropriate
therapy or dietary supplements in most cases.
c. Thyroid hormones have a permissive effect on other hormones, optimise sensitivity of
various tissues/systems, e.g. CNS, CVS and bone to these hormones.
d. The incidence of hypothyroidism is about 1:4 000-5 000 of newborns worldwide and
some countries screen for hypothyroidism in all newborns. This is because thyroid
hormones affect growth and development.
e. The thyroid gland weighs about 20 gm in adults and has tremendous capacity for
growth as seen in some goitres. Please note that thyroid gland can be enlarged in some
cases of hyperthyroidism and in hypothyroidism, e.g. due to iodine deficiency or
autoimmune disease with infiltration of thyroid gland.
Thyroid gland
Thyroid gland weighs about 20 g in adults and has tremendous capacity for growth as seen in
some goiters. The thyroid gland synthesises, stores and secretes thyroid hormones. It is derived
from the foregut as a down growth from the floor of the pharynx. The thyroid gland is made
up of 2 main lobes on either side of the trachea at the base of the neck. Within these lobes are
parafollicular or c-cells, which produce calcitonin and follicular cells (acni) that, produce
thyroid hormones; T4 and T3 as well as thyroglobulin. Please note that there are also parathyroid
glands nested within the thyroid gland and these synthesise and secrete parathyroid hormone
(see lecture of Ca2+ and HPO42- metabolism). The follicular cells contain a colloid substance
that is rich in thyroglobulin, a glycoprotein of 2 800 amino acid residues and MW of over 600
000 daltons. This protein, made up almost exclusively of tyrosine is the matrix in which thyroid
hormones are synthesized and stored. The thyroid gland is found in all vertebrates. There is no
recognizable thyroid gland in invertebrates but these animals synthesise thyroid hormones. The
thyroid gland is innervated by sympathetic and parasympathetic nerves whose main function
is to regulate blood flow and that controls TSH and iodine delivery. Two main blood supplies
to the thyroid are superior thyroid arteries from carotids and inferior thyroid arteries from
subclavians. Blood exits the thyroid via thyroid veins. The thyroid gland is highly vascularised
with blood flow that reaches 5 mL/g/min, which is twice that of kidney. When the thyroid gland
is enlarged, blood flow increases resulting in turbulent flow giving rise to an audible whistling
sound termed bruit. If the turbulence is palpable it is called thrill.
23
Figure: Thyroid gland (My Health Alberta – Government of Alberta)
Intake
Thyroid hormones have iodine in their structure and this means that lack of iodine in diet
can result in hypothyroidism. Iodine is ingested as organic or inorganic, which is converted to
inorganic iodide for absorption in the upper GIT and uptake by thyroid gland. Iodine intake
varies geographically and in many countries and iodine supplements cater for inadequacy in
diet. Deiodination of thyroglobulin and thyroid hormones also supplies the iodide pool in the
body. Iodine is efficiently absorbed by the GIT and little is lost in stool and kidney.
Trapping
The thyroid gland contains about 95% of body iodine. Iodide in blood is trapped against
concentration and electrical gradients by energy-dependent mechanisms possibly involving
Na+/K+ATPase, which establishes an inward-directed Na+ gradient. The actual transporter of
iodide is a Na+/I- cotransporter. The salivary, mammary and gastric glands can also trap iodine.
The trapping of iodine is stimulated by thyroid stimulating hormone (TSH).
Oxidation and organification
After transport or regeneration within the thyroid gland, iodide is oxidized to iodine by
membrane-bound peroxidase. This is followed by iodination of tyrosine of the thyroglobulin
also using peroxidase. When iodine covalently attaches to carbon compounds, this is called
organification. Iodination of tyrosine can occur at one or two places on the benzene ring to give
monoiodotyrosine and diiodotyrosine.
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Coupling
Monoiodotyrosine and diiodotyrosine are enzymatically coupled in 2 ways to give the 2 active
forms of thyroid hormones:
Diiodotyrosine + monoiodotryrosine: T3 (3,5,5-triiodotyrosine)
Diiodotyrosine + diioodotryrosine: T4 (3,5,3,5-tetraiodotyrosine)
These hormones remain attached to thyroglobulin in the colloid of follicular lumen.
Figure 8.1: Structures of thyroid hormones (Rae et al 2018)
Storage and release
The thyroid gland has the largest reservoir of any stored hormone in the body storing about 3
months’ supply of thyroid hormones, which are released slowly. This thyroid hormone
economy ensures continued supply of hormones into circulation should thyroid hormone
synthesis ceases. Thyroglobulin is the storage form of thyroid hormones in the thyroid gland.
Thyroid hormones are released in a process that first involves pinocytosis mechanism of colloid
in follicular cells to colloid droplets. This is followed by fusion of the droplets with lysosomes
to form phagolysocytic vacuole. In the vacuoles thyroglobulin is hydrolysed by proteases to
release into circulation T4 and T3 and perhaps small amounts of thyroglobulin. Proteolysis and
release of thyroid hormones are stimulated by TSH and inhibited by lithium and iodide.
Thyroglubulin fraction is also found in plasma but this fraction appears to come lymphatic
drainage and not from thyroid secretion directly into blood. The frequency of detecting this
25
fraction is increased in pregnancy, some thyroid tumours as well as in some cases of
hyperthyroidism. Increased release of thyroglobulin is also seen in inflammation such as in
thyroiditis. Please note that diiodotyrosine is also released from the thyroid while another small
fraction comes from cleavage of thyroid hormones in circulation. This diodotyrosine fraction
is decreased in hypothyroidism and increased in hyperthyroidism.
Figure 8.2: Synthesis and release of thyroid hormones (Rae et al 2018)
Figure: Synthesis and release of thyroid hormones (Austin Publishing group)
The above Figures have same information but differently presented.
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Peripheral metabolism of thyroxine (T4)
T4 can be deiodinated to give T3. Infact, >80% of circulating T3 is derived from T4. T4 can also
be deiodinated to give rT3 (3,3,5-triiodotyrosine). T3 and rT3 are further deiodinated to
T2. Although the thyroid gland is capable of deiodinating T4 to T3, the contribution of T3 in
circulation from this source is very small. Like other hormones, thyroid hormones in circulation
are bound to proteins for transport, conservation and storage.
Table: % fractions of thyroid hormones bound to proteins
Free
Thyroid binding globulin
(TBG)
Thyroid binding prealbumin
(TBPA)
Albumin
T4
0.04
70-75
25
10
T3
0.40
70-80
Trace
25-30
The half-life of TSH is 50-60 days, that of T4 is 7 days while T3 and rT3 have a half-lives of 2
and 1 days, respectively, TBG, an α globulin (MW 60 000) has a half-life of 5 days and TBPA
(MW 50 000) has a half-life of 2 days. TBG has a higher affinity for T4 than T3. Binding of T4
to TBPA is important when TBG is lacking. Bear in mind that although the percentages of free
fractions are low, it is always the free fraction that binds to receptors and therefore exerts
biological activity.
Since >70% of thyroid hormones are bound to TBG, it follows that the level of this protein is
a major factor in control of thyroid hormones. Drugs such as phenytoin, heparin and aspirin
compete with T4 and T3 for TBG-binding sites. Small seasonal variation in serum T4 and T3
levels are seen in some normals, the levels vary inversely with temperature.
Mechanism of action
Since thyroid hormones are small and lipophilic, they enter the cell. Unlike most steroid
hormones whose receptors are cytoplasmic, thyroid hormone receptors are in the nucleus.
Cytosolic binding proteins modulate entry of the hormones into nucleus. The receptors are
differentially expressed in different tissues. It is also thought that there exists a mitochondrion
receptor to control oxidative phosphorylation and there may also be cytoplasmic receptors.
Current evidence suggests that T3 has greater affinity for nuclear receptors than T4.
Control
Synthesis and release of thyroid hormones and TSH are controlled through feedback
mechanisms in the hypothalamic-pituitary-thyroid axis. TRH level is controlled by nervous
system and levels of T4 as well as T3. Low levels of T4 and T3 and TSH stimulate synthesis and
release of TRH. TRH acts in the anterior pituitary gland on thyrotrophs to release TSH and on
mammotrophs (lactotrophs) to modulate/increase release of prolactin. TRH is a tripeptide
(glutamyl-histidyl-proline), which is synthesized in hypothalamus from where it is transported
to the anterior pituitary gland. The release of TRH and TSH are episodic (pulsatile) and also
follows circadian rhythm with a decrease following onset of sleep. Release of T4 and T3 are
episodic and pulsatile.
TSH concentration is controlled by TRH and T4 as well as T3. High level of TRH and low level
of T4 and T3 stimulate synthesis and release of TSH. The release of TSH is a rapid response
27
while synthesis of TSH is a delayed response. The release of TSH like TRH is episodic
(pulsatile) and also follows circadian rhythm. In pregnancy, thyroid gland and BMR are
increased by about 25% and this is associated with elevated concentration of T4.
TSH acts in follicular cells in thyroid gland stimulating iodide uptake. Pumps, Na+/K+ATPase
(involved in iodine uptake by thyroid gland) is also stimulated by TSH. TSH stimulates
organification, coupling as well as proteolysis of thyroglobulin and release of T4 and T3 and
this is a rapid response while the slower response involves stimulation of thyroid growth. TSH
is a glycoprotein (MW 29 000) produced by thyrotrophs and it is made up of 2 subunits, which
are alpha and beta. Please note that LH, FSH and hCG are similar to TSH in that these 4
hormones have 2 subunits, which are alpha and beta. The alpha subunits are very similar with
respect to amino acid sequences but differ in CHO components while the differences lie in the
beta subunits. The beta subunits carry immunological and physiological characteristics of the
hormones.
Grave’s disease, is perhaps the commonest hyperthyroid disease and involves autoimmunity
with the patient making antibodies that bind to TSH receptor mimicking TSH. T4 and T3 levels
are high but TSH concentration is low and goiter also develops. On the other hand, Hashimoto’s
disease is a common autoimmune hypothyroid disease resulting in hypothyroidism. Unlike
Grave’s disease, which involves humoral immunity, Hashimoto’s disease is cell-mediated
immune attack of thyroid gland and eventually hypothyroidism develops due to destruction of
thyroid. It is not uncommon for these diseases to interchange in the same patient.
Function
All tissues are targets of thyroid hormones in varied ways and tissues that stand out include
liver, kidney, heart, brain, spleen and testes. Thyroid hormones basically control overall
metabolism in the body. On a weight basis, T3 is much potent (ten times) than T4 and is perhaps
the most influential on feedback mechanisms. The actions of T3 are also more rapid and of
shorter duration than T4.
Growth and development: Physiological levels of thyroid hormones stimulate protein synthesis
(rate of transcription and translation are increased) and therefore growth. These hormones can
be viewed as growth factors since no growth occurs in the absence of thyroid hormones even
if GH is present. Thyroid hormones stimulate release and increase effectiveness of growth
hormone (GH). Development of embryo is sustained in the presence of thyroid hormones.
Hypothyroidism is associated with reduced chances of a female getting pregnant as well as a
high loss of pregnancies. In pregnancy, the metabolic rate of the mother is elevated to meet
foetal needs, hence the mother is in a physiological hyperthyroid state. The elevated βhCG
(which is structurally similar to TSH) in pregnancy stimulates TSH receptors to synthesise and
release thyroid hormones.
Control of metabolism: Thyroid hormones stimulate BMR or calorigenesis and oxidative
phosphorylation in mitochondrion is increased by thyroid hormones.
CHO: Thyroid hormones increase intestinal absorption of glucose and galactose and
subsequent insulin dependent entry of glucose into adipose and muscle cells. The hormones
also increase CHO turnover by increasing glycogenolysis and gluconeogenesis.
28
Vitamins: Thyroid hormones are also required for synthesis of vitamin A from carotene and
for conversion of vitamin A to retinene, a pigment that is required for adaptation in dark. In
hypothyroidism, the level of carotene is elevated giving the skin a yellow tint, vitamin
deficiency may also occur.
Lipids: Turnover of lipids is increased by thyroid hormones. All aspects of lipid metabolism
i.e. synthesis, mobilisation and degradation are increased by thyroid hormones but the overall
effect is lipolysis resulting in decrease in fat storage. Thyroid hormones increase the sensitivity
of tissues to lipolytic effects of catecholamines, GH, glucagon, glucocorticoids in addition to
their direct effects on lipolysis. Hepatic synthesis of triglycerides is increased and T3 in
particular stimulates synthesis of LDL receptors and LDL degradation. Thyroid hormones
enhance cholesterol synthesis but this effect is overwhelmed by the effect of the hormones in
elevating the rates of faecal excretion of cholesterol and conversion of cholesterol into bile.
Thermogenesis/calorigenesis: Coupled to metabolism, thyroid hormones stimulate futile cycles
and this increases heat generation. Brain, spleen, pituitary and testes do not demonstrate
calorigenic response to thyroid hormones due to their temperature sensitive nature.
Erythropoiesis: Thyroid hormones increased RBC synthesis through increase in erythropoietin,
whose synthesis is increased as a result of increased O2 consumption. Furthermore, thyroid
hormones increase the level of 2, 3-DPG in RBCs to shift oxyhaemoglobin curve to the right
allowing release of O2 in response to tissue hypoxia. Some hypothyroid patients can therefore
suffer from pseudo or physiologic anaemia due to low 2, 3-DPG concentration as this reduced
the ability of RBCs to release O2 to tissue at any given haematocrit. In this situation, although
the concentration of RBCs and Hb are normal, O2 delivery is reduced and this mimics anaemia.
CVS: Thyroid hormones increase number and effectiveness of adrenergic receptors and this
affects CNS and CVS, skeletal muscle and adipose tissue. Thyroid hormones are ionotropic
and chronotropic on heart.
CNS: Lack of thyroid hormones especially in the 1st 6 months of life leads to retarded mental
development. It appears that thyroid hormones are crucial for myelinisation and proliferation
of neural cells. Thyroid hormones maintain respiratory center in the brain. Hence,
hypothyroidism can result in respiratory failure especially in patients with an underlying lung
disorder.
Thyroid hormones and sexuality and reproduction
Thyroid disease is more common in females than in males. In females, goiter is common in
puberty, pregnancy and menopause. In pregnancy, thyroid gland is enlarged with increased
blood flow and a bruit may be present. Total T4 and T3 levels in plasma are increased due to
elevation in TBG level as a result of increased oestrogens. The increase in T4 concentration is
greater than that of T3. BMR increases by as much as 20-30% by term and this is due to increase
in body mass.
29
Hormone deficiency
Hypothyroidism is caused by:
1O: Thyroiditis, e.g. Hashimoto disease.
Therapeutic ablation
Iodine therapy
Surgery.
Thyroid dysgenesis
Aplasia
Dysplasia.
Abnormal hormonogenesis
Iodine deficiency
Iodine excess
Thyroid blocking drugs
Thyroglobulin metabolism defects
Lack of peroxidase and or H2O2
Reduced coupling.
2O: Pituitary defects (TSH deficiency).
Reduced peripheral response to thyroid hormones.
Thyroid hormone deficiency in infants causes cretinism. Mental development and growth are
retarded and this is coupled to delay in sitting, walking and talking. Impairment of linear
growth leads to dwarfism that is characterised by limbs which are disproportionately short
when compared to the trunk. If hormone deficiency is in late childhood, mental retardation is
less prominent and impairment of linear growth is a major feature.
Symptoms of thyroid hormone deficiency in adults appear over months or years. These
symptoms are non-specific but include tiredness, lethargy and constipation. Mental function
and motor activity are also decreased. These symptoms are due to decreased β adrenergic
activity and sensitivity, as well as decreased BMR. Reduced BMR is also responsible for cold
intolerance, increased body weight and sometimes obesity. Increased body weight and
obesity can lead to reduced appetite. Decreased protein synthesis and accumulation of
mucopolysaccharides in subcutaneous tissue are associated with myxoedema. Dermal
infiltration can be seen resulting in thickness of feet and hands that do not respond to
pressure.
30
Menstruation is characterised by increased blood flow and in part this leads infertility. The
skin is dry and coarse due to low BMR and decreased activity of futile cycles. Hoarse voice,
hair loss and brittle nails are common and very noticeable in females. Decreased metabolism
of vitamins might result in reduced conversion of carotene to vitamin A; this leads to the skin
becoming jaundice-like.
Since thyroid hormones increase the number and effectiveness of beta adrenergic receptors in
the CNS and CVS, reduced hormone concentration can lead to bradycardia. Decreased
cardiac output and heart rate increases circulation time. Ultimately the heart enlarges, which
may lead to failure. Reduced effectiveness of β adrenergic reception perhaps leads to reduced
GIT motility and constipation, leading to weight gain. Decreased mental function is
associated with reduced memory, speech, initiative, etc. T3 prevents smooth muscle
contraction and this might explain in part why some patients with hypothyroidism experience
hypertension.
Conditions such as chronic carbohydrate deprivation, starvation, under nutrition and
malnutrition, anorexia nervosa, diabetes mellitus, liver and renal diseases, myocardial
infarction and systemic illness are associated with reduced conversion of T4 to T3. In these
conditions, plasma T3 is low but total T4, freeT4 and TSH are usually normal and rT3 may be
elevated due to reduced clearance: low T3 syndrome or euthyroid sick syndrome. This
syndrome reduces metabolic rate to conserve energy and nitrogen as an adaptation to stress.
Please note that illness in general tends to decrease TSH secretion by the pituitary gland and
the mechanism is not clear. In conditions such as liver and renal failures, concentration of
hormone binding proteins is reduced due to reduced synthesis and increased urinary loss,
respectively.
Hormone excess
Hyperthyroidism is caused by:
1O: Thyroiditis, e.g. Hashimoto disease.
Iatrogenic.
Thyroid adenoma and carcinoma.
2O: Pituitary tumours (TSH excess).
3O: Hypothalamic tumours (TRH excess).
Thyroid stimulating antibodies or thyroid receptor antibodies e.g. in Grave’s disease.
Thyrotoxicosis and hyperthyroidism are used interchangeably, but strictly thyrotoxicosis is a
biochemical/physiological phenomenon that results when tissues are presented with excessive
thyroid hormones. These hormones need not necessarily came from the thyroid gland as in
hyperthyroidism.
Although physiological levels of thyroid hormones stimulate protein synthesis, increased
concentration of the hormones accelerates protein catabolism, leading to increased N2
excretion. Coupled to this, increased BMR results in weight loss – leading to increased food
intake, which can normalise weight or increase weight. These patients have high basal
metabolism and together with stimulation of futile cycles leads to heat sensitivity,
perspirations and weight loss. The moist skin is due to vasodilatation as a result of increased
31
temperature and telangiectasia (weblike dilated blood vessels) is a feature.
The patients can present with restlessness, over anxiety, exaggerated reflexes and irritability
perhaps due to the fact that thyroid hormones increase the number and effectiveness of β
adrenergic receptors affecting CNS.
This effect on β adrenergic receptors in the CVS can lead to tachycardia resulting in feeling
of pounding of heart. Increased β adrenergic activity and sensitivity is also responsible for
palpitations. Increased blood pressure and cardiac failure can also occur. The increased
cardiac output and heart rate reduces circulation time, hence, shortness of breath. This
coupled to increased N2 excretion results in muscle wasting, including muscles of the
respiratory system and general weakness leaving these patients with dyspnoea (laboured
breathing) and difficulties in rising from sitting or squatting. Muscle weakness is also due to
the inability of muscle to phosphorylate creatine in hyperthyroidism; creatinuria is therefore a
feature. Increased effectiveness of β adrenergic reception perhaps leads to increased GIT
motility, and this results in hyper-defaecation and diarrhoea, contributing to weight loss.
Hyperthyroid patients have enhanced intestinal glucose absorption. Furthermore, excess
thyroid hormone level stimulates adrenaline-induced glycogenolysis, decreasing hepatic
glycogen store. Insulin degradation is enhanced by thyroid hormones augmenting other
factors that elevate plasma glucose in thyrotoxicosis. Decreased plasma cholesterol level is
also a feature due to increased degradation.
Menstrual flow can be reduced or absent.
Excess thyroid hormones increase metabolism of a number of drugs, hence the half-life of
these drugs is reduced. Therefore, in hyperthyroid patients, the usual dose of the drug might
be insufficient. Please also note that excess thyroid hormones may in some cases increase
activity of certain drugs, even though the half-life of these drugs is reduced. This is due to the
fact that thyroid hormones increase the number of some receptors in tissues (The converse
holds in low thyroid hormone states.)
Increased thyroid hormones level stimulates bone resorption. This leads to bone loss,
hypercalcaemia, calciuria, phosphaturia and urinary loss of collagen if the situation persists.
Hypercalcaemia may lead to renal failure. Osteoporosis and fractures can be complications in
hypothyroid mature women who are over prescribed thyroid hormones. Anorexia, nausea and
vomiting are thought to be in part due to hypercalcaemia if hyperthyroidism is severe.
Excess thyroid hormone levels reduce the synthesis and activity of superoxide dismutase, an
enzyme involved in antioxidant activity. Hence, hyperthyroidism increases exposure to
oxidant damage.
Autoantibodies against parietal cell are seen in some patients with Grave’s disease and this
may result in anaemia.
32
Goitre
Goitre is the enlargement of thyroid gland and can be seen in hypo and hyperthyroidism and
it is caused by:
a. Excessive stimulation of the thyroid gland by TSH in an attempt to normalise thyroid
hormone levels e.g. in iodine deficiency.
b. Antibodies stimulating growth of thyroid gland e.g. in Grave’s disease.
c. Inflammation of the thyroid gland and infiltration by lymphocytes e.g. in Hashimoto’s
disease.
d. There are times when there is a small focus in the gland that is growing
autonomously. If it is hyper secreting thyroid hormones it is toxic nodular goitre; if
not it is a cold nodule.
Grave’s disease, is perhaps the commonest hyperthyroid disease and involves autoimmunity
with the patient making antibodies that bind to their TSH receptor mimicking TSH. T4 and T3
levels are high but TSH concentration is low and goiter also develops.
Biochemical investigation of thyroid gland disorders
Blood concentration of hormones and binding proteins
Total T4 and T3.
Free T4 and free T3.
TSH, TRH.
Thyroglobulin.
TBG.
Thyroid antibodies: e.g. anti-thyroid peroxidase and thyroid receptor antibodies
Table of how TSH, T4 and T3 appears in hypothyroidism
Disorder
Plasma [TSH]
Plasma [T4]
Plasma [T3]
1O (Thyroid)
Elevated
Reduced
Reduced
2O (Pituitary)
Reduced
Reduced
Reduced
3O (Hypothalamus)
Reduced
Reduced
Reduced
Metabolic impact of thyroid hormones
Lipids: cholesterol, triglycerides.
BMR.
Glucose
Ca2+, HPO42-.
Liver, cardiac and muscle function tests.
33
Dynamic tests
TRH test: Administration of TRH (200 μg i.v.) in normal subjects results in elevation of TSH
by 5-fold after about 25 minutes and returns to baseline after 3½ hours. In the protocol, blood
is taken at 0, 20 and 60 minutes after TRH administration. Please note that females respond
more by comparison with males and in females the response is greater in the follicular than
the luteal phase. The response to TRH can also decline with age.
Example of interpretation of TRH stimulation test in hypothyroidism
Disorder
Plasma [TSH]
Plasma [T4]
Plasma [T3]
1O (Thyroid)
Elevated
Reduced
Reduced
2O (Pituitary)
Reduced
Reduced
Reduced
3O (Hypothalamus)
Elevated
Elevated
Elevated
TSH test: This test distinguishes 1O from 2O hypothyroidism. In both cases T4 and T3 are low.
Administration of TSH increases T4 and T3 in 2O hypothyroidism but not in 1O
hypothyroidism. This test is not useful as TSH can be measured directly.
34
Topic 4 Adrenal cortex hormones dysfunction
Rae P., Crane M., & Pattenden R. (2018). Clinical Biochemistry: Lecture
notes (10th ed.), Chapter 9. Wiley-Blackwell.
Assumed previous knowledge
Biochemistry
 General structure of steroids
 Functions of hormones
Human Anatomy and Physiology / Physiological Sciences
 How endocrine organs work
 Location and structure of the adrenal glands
Learning outcomes
 describe structure of adrenal cortex including the morphological zonation;
 classify adrenocortical hormones and outline the synthesis of adrenal steroids from
cholesterol and their transport;
 describe the structure, regulation and clinical significance of corticotrophin (ACTH);
 analyse control of glucocorticoid hormone secretion, in terms of hypothalamusanterior pituitary-adrenal gland axis;
 describe the functions of glucocorticoids;
 analyse pathological conditions affecting adrenocortical function, including;
(a) range of disorders resulting in hypofunction
(b) range of disorders resulting in hyperfunction
 evaluate the tests for adrenocortical function, including:
(a) serum cortisol measurements
(b) serum ACTH (corticotropin) tests
(c) stimulation and suppression tests
(d) other tests in management of patients with adrenocortical dysfunction
35
Normal adrenal cortical function
The adrenocortical gland produces a number of hormones: glucocorticoids,
mineralocorticoids and androgens. Mineralocorticoid (aldosterone) was also covered in water
and salt regulation in BMS207. Sex hormones (androgens) will also the covered in
reproductive endocrinology disorders.
A number of steroid hormones are synthesised in the adrenal cortex and these include
glucocorticoids, which are important physiologically and clinically as well as
mineralocorticoids that are important physiologically. Structurally the adrenal cortex is made
up of three distinct layers; the zona glomerulosa (outermost), zona fasciculata and zona
reticularis (innermost). The zona fasciculata synthesises glucocorticoids (of which cortisol is
the most important in humans) and a little bit of androgens, the zona glomerulosa synthesises
aldosterone, the principal mineralocorticoid and the zona reticulosa synthesises mainly
androgens and a little bit of cortisol.
Steroid hormone structure
All steroid hormones are derived from cholesterol and contain the
cyclopentanoperhydrophenanthrene nucleus (cholesterol ring structure). Understanding the
structure and numbering system used in the cholesterol nucleus is important as it helps
understand steroid biochemistry. Cholesterol, the parent steroid, contains 4 rings plus a
carbon side-chain as shown in the reading: it contains 27 carbon atoms. The important parts
of the steroid ring system in determining steroid hormone function are:
 The A ring, which is aromatic.
 Carbon atom 11, which is hydroxylated in cortisol, corticosterone and aldosterone.
 Carbon atom 17, which has a hydroxyl group attached in cortisol, 11-deoxycortisol,
testosterone and oestradiol, a keto group attached in oestrone and some androgens.
 The side chain of carbon atoms 20 and 21, which is present in progesterone,
glucocorticoids and aldosterone, but absent in androgens and oestrogens.
Refer to biochemistry and physiology texts for general structure of steroids
The adrenal cortex secretes 3 types of steroids in each of the 3 zones and the steroids can be
classified according to the number of carbon atoms (C-18: oestrogens, C-19: androgens and
C-21: aldosterone, cortisol and corticosterone) and can also be classified according to their
major effects:
a. Glucocorticoids:
The major glucocorticoid in humans is cortisol.
Small amounts of corticosterone, which is a weak glucocorticoid are also secreted.
b. Mineralocorticoid:
The primary mineralocorticoid is aldosterone.
Small amounts of deoxycorticosterone are also secreted.
c. Sex steroids:
The major androgen is dihydroxyepiandrosterone, which is mainly sulphated.
The other androgens are androstenedione and 11β-hydroxyandrostenedione.
36
Steroid hormone synthesis in the adrenal cortex
Adrenal steroid hormones are synthesised from cholesterol shown in Figure 9.5 in prescribed
text. This pathway is important, since it explains some of the biochemical effects of
abnormalities in glucocorticoid secretion and understanding this pathway is the basis for
knowing the complications of adrenocortical diseases.
Cholesterol is synthesised from acetate or can be taken up from circulating LDL particles.
Conversion of cholesterol to pregnenolone is rate limiting especially in the absence of stress.
This conversion is mainly controlled by ACTH, angiotensin 11 and K+ in the glomerulosa
and by ACTH in the fasciculata and reticularis. The pathway from cholesterol to
pregnenolone is common to all steroid hormones including those of gonadal origin. The main
biosynthetic pathways for the synthesis of the 3 types of adrenal steroids from pregnenolone
are shown in Figure 9.5. The cortical zones contain different enzymes and this determines the
steroid synthesised. There are 2 pathways for synthesis of cortisol: Δ5 pathway via 17
hydroxypregnenolone, which is the predominant pathway in humans and the Δ4 pathway via
progesterone. The consequence of 3 different but interlocked pathways using the same
precursor has clinical implications. For instance, in 21 -hydroxylase deficiency, cortisol level
is low and the intermediates before blockage are therefore elevated and are moved into
androgen synthesis, which is elevated.
Steroid biosynthesis in adrenal cortex (Figure 9.5 of prescribed text)
37
Figure: Adrenocortical hormone biosynthesis showing mutated enzymes that result in
CAH (Pathway Medicine)
Cortisol (Glucocorticoid)
Glucocorticoid transport and catabolism
Unlike polypeptides and catecholamines, adrenal steroids are not stored but are secreted into
blood immediately after synthesis. Steroids in blood are free and to a large extent bound to
proteins for circulation and to increase ½ life. All steroids bind to albumin to varying extents
but some steroids have specific binding proteins: cortisol is transported by cortisol binding
globulin and androgens and oestrogens are transported by sex hormone binding globulin.
These binding proteins also bind other steroids but with reduced affinity. Bound steroids tend
to be inactive.
Steroids are catabolised in the liver and the products are then conjugated to glucuronate.
These compounds enter the circulation and are excreted in urine. Intact hormones are also
excreted in small quantities.
Receptors and mechanism of action
Steroids are small and lipid soluble, hence penetrate membranes and transverse into
cytoplasm where there are receptors. The receptors are cytosolic and perhaps also nuclear.
The distribution of receptors varies depending on function of hormone. Glucocorticoid
receptors are found in a wide variety of cells, consistent with their function.
38
Individual steroids in addition to high affinity for their specific receptors have some affinity
for receptors of other steroids. Hence, increased cortisol, which is a glucocorticoid, results in
spill-over into aldosterone (mineralocorticoid) receptors and this manifests as
mineralocorticoid excess resulting in Na+ retention, K+ loss and hypertension.
Actions of glucocorticoid hormones
Physiological/pathophysiological
Carbohydrates: Glucocorticoids have important effects on carbohydrate metabolism, acting in
opposition to insulin to raise concentration of blood glucose levels so that glucose is supplied
to tissues such as brain. The increased glucose level comes via gluconeogenesis, in which
precursors of glucose are made available to the liver from muscle and adipose tissue. Cortisol
enhances the release of amino acids from proteins in muscle and other tissues. There is also
an increase in activity of liver enzymes involved in gluconeogenesis.
Protein: The overall effect of glucocorticoids on protein metabolism is catabolic.
Glucocorticoids cause a negative nitrogen balance and loss of protein from tissues such as
muscle and bone. The amino acids released are mobilised to the liver and used in synthesis of
protein and in gluconeogenesis.
Lipids: Glucocorticoids increase lipolysis and at the same time decreasing fatty acid
synthesis. The resultant FFAs are channelled into gluconeogenesis. In extremities,
glucocorticoids promote lipolysis while in abdomen glucocorticoids promote fat deposition.
Fat is therefore distributed from the periphery to the centre, neck and face.
Immunity: Large doses of glucocorticoids suppress immune system with the effect being
more pronounced on cellular than humoral immunity. Glucocorticoids depress T-cell
proliferation, reduce synthesis of complement and immunoglobulins such as IgG and increase
B- and T-cell destruction. Immature and less fully differentiated immune cells are not
affected much by toxic effects of glucocorticoids. This is beneficial to patients on
glucocorticoid therapy since discontinuation of therapy reverses suppression of immunity.
Glucocorticoids are anti-inflammatory and this property is through inhibition of
phospholipase A2 by glucocorticoids.
Electrolytes: Glucocorticoids have mineralocorticoid activity. Although cortisol causes
retention of Na+ and H2O and excretion of K+ in the kidney, this effect is negligible compared
to that of aldosterone in physiological conditions. However, these effects are enhanced due to
spill-over if cortisol is in large amounts.
CVS: Glucocorticoids augment the effect of catecholamines on peripheral vessels. The
resultant increased peripheral resistance coupled with Na+ and H2O retention increases blood
pressure.
Gastrum: Excessive amounts of cortisol block prostaglandin synthesis and decrease mucus
secretion. In addition, there is increased secretion of pepsin and HCl resulting in increased
liability to peptic ulceration.
Bone, Ca2+ and HPO4-: Glucocorticoids inhibit osteoblastic and increase osteoclastic
activities. Glucocorticoids antagonise the action of vitamin D3 in GIT and kidney. The latter
39
effect coupled to increase in GFR by glucocorticoids results in urinary loss of Ca2+ and
HPO4-. Glucocorticoids inhibit synthesis of collagen and other matrix proteins thereby
reducing bone matrix formation. Therefore, chronic effect of glucocorticoids on bone leads to
osteoporosis. The interference by glucocorticoids on matrix protein synthesis accounts for
easy bruising, abdominal striae, muscle weakness and poor wound healing in patients with
glucocorticoid excess.
Reproduction: Excess glucocorticoids reduce the capacity of male and female reproductive
functions and LH and FSH are low. This is perhaps an attempt to delay reproduction during
stress.
Growth and development: Excess glucocorticoids interfere with development and inhibit
linear growth as seen the children with Cushing’s disease. Therefore, administration of
glucocorticoids to children should be done with caution.
Others:
Increased glucocorticoid levels appear to be associated with jet lag, insomnia, euphoria,
increased appetite and lowered sensory threshold.
Reduced glucocorticoids levels are associated with depression, lethargy, apathy and failure to
concentrate.
Due to reasons that are not clear, there is increased chances of cataract formation and
intraocular pressure in patients on exogenous glucocorticoids but these effects are not
observed in patients with endogenous elevated glucocorticoids.
Pharmacological
Glucocorticoids are widely used in therapy to treat conditions such as rheumatoid arthritis,
lupus erythematosus, rheumatic fever, some leukaemias, asthma, allergies and suppression of
transplant rejection.
Anti-inflammation/anti-allergic effects: Glucocorticoids inhibit inflammatory response to
infection and injury. This is because cortisol reduces capillary dilatation, reduces
prostaglandin and leukotriene synthesis (by inhibiting phospholipase A2, which catalyses
formation of arachidonic acid, a precursor of prostaglandins and
leukotrienes). Glucocorticoids inhibit synthesis and release of histamine. Glucocorticoids also
decrease leucocyte migration and stabilise lysosomal membranes.
Suppression of immunity: Glucocorticoids depress T-cell proliferation, reduce synthesis of
complement and immunoglobulins and increase B- and T-cell destruction. Lymph node and
thymus sizes are reduced by glucocorticoids. Hence, glucocorticoids are used in treatment of
lymphomas and in management of transplants.
Control of glucocorticoid secretion: the hypothalamus–pituitary gland–adrenal cortex axis
Unlike other hormones, adrenocortical hormones are not stored in the adrenal gland. These
hormones are synthesised and release on demand and this is rapid. The steroids secreted by
fasciculata and reticularis are principally under control of corticotrophin or
adrenocorticotrophic hormone (ACTH) while renin-angiotensin system controls secretion
from glomerulosa. In other words, cortisol, corticosterone, 18-OH corticosteroids and
androgens are regulated by ACTH from anterior pituitary gland while the renin-angiotensin
40
system regulates aldosterone synthesis and secretion. Stimulation of androgens by ACTH
means that in diseases such as Cushings’, androgen secretion is also elevated.
ACTH is a single polypeptide of 39 amino acids. The biologically active part of ACTH
molecule is its N-terminal 26 amino acids; this fragment is almost equal to the complete
molecule in biological potency. ACTH has a very short circulating ½ life of less than 10
minutes. ACTH stimulates a number of rate limiting steps in steroid synthesis in adrenal
gland and increases blood flow to the adrenal medulla and secretion of synthesised steroids.
In addition, ACTH stimulates hypertrophy and hyperplasia of adrenal gland.
The secretion of ACTH is in turn controlled by the hypothalamus, which releases
corticotrophin-releasing hormone (CRH) that is secreted by neurones of the paraventricular
nucleus. CRH stimulates pro-opiomelanocortin synthesis in corticotroph cells of anterior
pituitary gland. Pro-opiomelanocortin is then processed to ACTH and other peptides.
To understand the control of adrenal steroids secretion, one must know the stimuli, which
releases CRH: corticosteroids themselves exert negative feedback control on CRH-ACTH
secretion. Secretions of ACTH and of CRH are episodic and the frequency and duration of
the episodes cause a circadian rhythm in secretion and circulating level of cortisol.
Superimposed on these 2 regulators of ACTH secretion is stimulatory influence of
stress. Regulation of ACTH secretion by normal pituitary gland is therefore governed by 3
factors:
(a). feedback action of cortisol-like steroids
(b). pituitary/adrenal rhythms (biological clock)
(c). stress
Pituitary-adrenal rhythms: The normal human has higher blood ACTH level hence higher
cortisol concentration in the morning (around 8am) than at night (around 12 midnight) and
shows a number of secretory episodes of these hormones during a 24-hour period. Subjects
such as security guards and health personnel who work at night and sleep during the day have
reversal of this pattern. This is important to note especially in Chemical Pathology when
cortisol results need interpretation. The circadian rhythm develops at the age of one year.
41
Figure: Biological clock of cortisol secretion (Integrative Therapeutics)
Stress: Superimposed on all other regulators of ACTH secretion is stimulatory influence of
stress (physical and psychological). Regardless of level of plasma cortisol, the normal
individual responds to major stress with a brisk increase in ACTH secretion and a consequent
increase in cortisol secretion. The greater the stress the higher the level of cortisol secreted.
The effect of acute stress on cortisol secretion is significant since chronic stress is
accompanied by adaptation. Among stresses, which induce increased pituitary/adrenal
activity are trauma, acute hypoglycaemia, acute anxiety, infections and immune reactions.
Stress and diurnal rhythm are important in cortisol secretion and can override negative
feedback loop.
Aldosterone
This was covered in BMS207: Clinical Biochemistry 1.
Aldosterone is the major mineralcorticoid produced in humans and acts along with other
steroids with mineralocorticoid activity to increase reabsorption of Na+ from urine, sweat,
saliva and gastric secretions. Since Na+ ions are exchanged for K+ and H+ ions, the
reabsorption of Na+ can result in K+ loss. ACTH has a minimal effect on aldosterone
secretion and the principal control seems to be the renin-angiotensin system.
Adrenal gonadal steroids
Under normal circumstances only small amounts of sex steroids are produced by the adrenal
glands. However, under certain pathological conditions, production of these hormones can
become important.
42
Pathology of the adrenal cortex/evaluation of adrenocortical function
Adrenocortical hyperfunction
The hyperfunctioning adrenal cortex can produce syndromes of hypercortisolism,
mineralocorticoid excess or virilism.
What are Cushing’s syndrome and Cushing’s disease?
Cushing’s syndrome versus Cushing’s disease (often used interchangeably)
Cushing’s syndrome occurs when there is excess cortisol in the body, regardless of the source
and cause. When the source of excess cortisol is pituitary then it is called Cushing’s disease.
Cushing’s syndrome (excess cortisol secretion) The features of Cushing’s syndrome are due
to the biochemical action of cortisol.
Abnormal circadian rhythm: Patients with Cushing’s syndrome lack the normal diurnal
rhythm in cortisol secretion. Normals show lower plasma cortisol level in the evening than in
the morning. In Cushing’s syndrome, these levels are not markedly different. The levels
might not necessarily be outside the reference range.
Urinary free cortisol: Urinary free cortisol excretion is a direct reflection of unbound cortisol
in plasma. This fraction is raised in almost all cases of Cushing’s. 24-hour urine collection is
ideal for urine cortisol measurement. Urine cortisol can also be expressed/urine creatinine,
with creatinine as internal standard.
Salivary cortisol can be measured (from page 126 of prescribed text).
Dexamethasone suppression test (page 126 of prescribed text): dexamethasone is a synthetic
glucocorticoid that is 25 times more potent than cortisol. It acts as a powerful feedback
inhibitor of ACTH secretion by binding to glucocorticoid receptors in anterior pituitary gland
thus interrupts the hypothalamic anterior pituitary axis. It can be used in the low dose format
as a screening test or in the high dose suppression test format to ascertain the origin of
adrenocortical hyper-function.
After administration of dexamethasone orally, cortisol in urine or blood or saliva is measured
after a defined period. Suppression is observation of a fall of cortisol of <50% of basal level
and this is seen in normals. This fall is due to suppressing effect of dexamethasone on ACTH
secretion and cortisol secretion. In the majority of patients with Cushing’s syndrome, cortisol
level does not significantly fall. ACTH levels can also be measured and these are reduced in
normals in response to dexamethasone.
Adrenocortical hypofunction (Addison’s disease)
Pituitary abnormality resulting in low secretion of ACTH leads to 2O adrenocortical
insufficiency.
1O adrenocortical insufficiency is due to destruction of adrenal cortex. Regardless of the
underlying process this leads to Addison’s disease.
The major clinical manifestations of adrenocortical hypofunction are attributable to
deficiency of aldosterone, cortisol and excessive ACTH production.
43
ACTH stimulation test (page 132 of prescribed text): The main biochemical finding in
patients with adrenal hypofunction is a failure of plasma cortisol level to rise rapidly i.e.
within 30 minutes in response to ACTH injection i.m., although the unstimulated level of
such patients may not be outside the normal range. In normal subjects, plasma cortisol level
rises within 30 minutes of ACTH injection. Tetracosactrin or synacthen (a synthetic analogue
of ACTH comprising the N-terminal 24 amino acids) is normally used) and it stimulates the
adrenal gland directly.
Adrenal hyperplasia
Congenital adrenal hyperplasia is an example of a genic defect affecting the synthesis of
glucocorticoids; briefly, the effects of an enzymic defect are two-fold:
 deficiency of the final product, in these cases cortisol and aldosterone;
 build up of intermediates before the blockage and diversion of these into other
pathways.
Note that the pathway of synthesis of androgens leads off from 17-hydroxy-pregnenalone.
Thus in these conditions, the adrenal cortex turns over to synthesising androgens with
virilising effects. Significant enzyme defects have been identified and include, 21-
hydroxylase and 11-hydroxylase.
CAH is an inherited deficiency of one of the enzymes in biosynthesis of cortisol, most
commonly the 21-hydroxylase. This results in compensatory increase in ACTH secretion,
which may be sufficient to keep plasma cortisol within normal range.
The increased stimulation of adrenal by ACTH causes the gland to be hyperplastic. Hence,
the name ‘congenital adrenal hyperplasia’. Increased stimulation of adrenal by ACTH also
results in increased formation of steroid precursors up to the point at which enzyme block
occurs.
Some of the precursors and their by-products have biological activities, which assume
clinical importance when excreted in excessive amounts even though they are of little clinical
significance when excreted in normal amounts. Deficiency in 21-hydroxylase results in
overproduction of androgens.
Clinical features of congenital adrenal hyperplasia
Androgens: The excessive androgen production may:
a. Affect genitalia of a female foetus, causing baby girl have ambiguous genitalia.
b. Cause precocious puberty in male child.
c. Cause hirsutism, 1O amenorrhoea and other signs of virilism in female.
The severity of symptoms depends on extent of enzyme deficiency.
Salt-losing crisis: Some infants with 21- hydroxylase deficiency present with salt-losing
crisis. A possible explanation why some but not all patients show salt-losing symptoms is that
there are 2 different 21-hydroxylase enzymes, one for 17-hydroxysteroids and the other for
11-hydroxysteroids.
44
A deficiency of 21-hydroxylase for none 17-hydroxysteroids can decrease mineralocorticoid
synthesis, hence salt-losing syndrome. The other explanation is that salt-losing form of the
disease is associated with more severe form of the deficiency.
Summary of laboratory evaluation of adrenocortical function
 immunoassays of blood cortisol & ACTH levels;
 most samples for cortisol determination are collected early in the morning when levels
are maximal;
 salivary cortisol gives good reflection of the free hormone;
 urinary cortisol levels give a good estimate of the amount of free cortisol in plasma,
which is an important screening test for adrenocortical hyperfunction;
 loss of the rhythm for cortisol is an important diagnostic tests for adrenocortical
hyperfunction;
 plasma ACTH measurements are important in diagnosing the cause of both
adrenocortical hyper- and hypo-function;
 dynamic test (dexamethasone suppression test and synacthen stimulation test);
 assessment of metabolic effects of cortisol: glucose, electrolytes and water balance,
lipids, proteins, GFR, acid-base, etc.
To re-cap: glucocorticoid stimulation and suppression tests (detailed in prescribed text)
You should understand the role of the various stimulation and suppression tests, in particular
the way in which these agents act at different points of the hypothalamus-pituitary-adrenal
cortex axis.
Glucocorticoid stimulation tests
These are largely (although not exclusively) used to assess hypofunction; stimulate the axis
and there is normally a response involving increasing plasma cortisol production:
 tetracosactrin is a synthetic analogue of ACTH which stimulates the adrenal gland
directly. Note that this can be used in the short format as a screening test or in the
longer format to ascertain the origin of adrenocortical hypofunction;
 CRH stimulation stimulates release of ACTH by the anterior pituitary.
Glucocorticoid suppression tests
In contrast to stimulation tests, these are used to assess hyperfunction; suppress the axis and
there is normally a response involving decreasing plasma cortisol production:
 dexamethasone or betamethasone acts as a powerful feedback inhibitor on the
hypothalamus. It can be used in the low dose format as a screening test or in the high
dose suppression test format to ascertain the origin of adrenocortical hyperfunction.
45
Other tests
Hypothalamic/Pituitary function (hormones)
Electrolytes and acid/base
Glucose
Lipids
46
Topic 5 Reproductive endocrinology dysfunction
Rae P., Crane M., & Pattenden R. (2018). Clinical Biochemistry:
Lecture notes (10th ed.), Chapters 10 & 11. Wiley-Blackwell.
Assumed previous knowledge
Human Anatomy and Physiology/Physiological Sciences
 Structures of the male and female reproductive organs
 Physiology of the reproductive system; male and female
Learning outcomes
 outline the steps in synthesis of female and male reproductive steroids from
cholesterol;
 describe the structures and functions of male and female reproductive hormones;
 analyse control of hypothalamus-anterior pituitary-testis/ovarian axes;
 analyse pathological conditions affecting male and female reproductive functions and
evaluate tests for reproductive function.
Introduction
In males and females, the gonads have exocrine function (production of gametes) and
endocrine function (production of hormones). There are similarities in the control of these
activities (hypothalamo-anterior pituitary-gonadal axis), but there are also differences. The
hypothalamus controls the release of gonadotropins (LH: luteinising hormone and FSH:
follicle stimulating hormone) via gonadotrophin releasing hormone (GnRH, also known as
LHRH: luteinising hormone releasing hormone). The testes respond to FSH by producing
sperm and to LH by producing testosterone. The ovaries respond to FSH with follicular
development and oestradiol production and to LH with ovulation, formation of a corpus
luteum and secretion of progesterone.
Genetic sex determination
In humans, sexual genotype is determined by 2 of the 46 chromosomes at conception. The XX
chromosomes give a genetically female while XY chromosomes result in male. The embryo is
sexually indifferent initially and the Y chromosome is responsible for making the indifferent
foetal gonads develop into testes and in absence of Y chromosome, gonads develop into
ovaries. Although X chromosome does not determine genetic sex of an individual, it plays a
role in sexual development since it carries some genes, which are essential for development of
males and females.
47
Reproductive development in males and females
Foetal testis synthesises 2 hormones; mullerian-inhibiting hormone (MIH) from Sertoli cells
and testosterone from Leydig cells. These 2 hormones determine sexual phenotype and are
secreted shortly after foetal testis forms. Leydig cells are mainly controlled by LH while FSH
mainly controls Sertoli cells. Sertoli cells also produce inhibin that negatively-feedbacks on
anterior pituitary gland in response to FSH. LH and FSH are also controlled by GnRH from
the hypothalamus.
An intact hypothalamic-anterior pituitary axis is important for full development of male
reproductive system, spermatogenesis and fertility. MIH and testosterone then transform the
indifferent urogenital tract, which at this time is made of primordial genital ducts from male
(wolffian duct) and female (mullerian ducts) into one that is male in character. In other words,
male reproductive tract is derived from wolffian ducts while female reproductive tract (oviduct
and uterus) is derived from mullerian ducts. Both ducts are present before sexual differentiation
and therefore the embryo has the potential to develop into either male or female.
The wolffian ducts give rise to various structures of male reproductive system including
prostate, epididymis and seminal vesicles, penis and scrotum. These processes are stimulated
by testosterone. Virilisation of external genitalia requires that testosterone be converted first
to dihydrotestosterone (DHT) by 5 alpha reductase. In females, lack of testosterone from
ovary leads to regression of wolffian ducts and external genitalia develop into clitoris, labiae
majora and manora. Mullerian ducts develop into oviducts due to absence of MIH.
Sexual phenotype differentiation is completed in about 14 weeks in males and 10-20 weeks in
females. There is very little/no contribution by LH from anterior pituitary gland in the control
of testosterone instead hCG from placenta stimulates testosterone secretion. During foetal
development, maternal hCG (human chorionic gonadotrophin), which is structurally similar
to LH, acts on the foetal testis to cause Leydig cell production of testosterone, which is
essential for normal development of male sex organs.
After foetal development testosterone negatively feedbacks on anterior pituitary gland to
inhibit LH secretion and on the hypothalamus to inhibit release of GnRH. FSH acts primarily
on the Sertoli cells (synergistically with testosterone) to allow spermatogenesis to proceed.
FSH production is controlled by GnRH, other peptide hormones (inhibin and activin)
produced by the Sertoli cells specifically inhibit and stimulate FSH production, respectively.
In circulation about, 60% of testosterone is bound to sex hormone binding globulin (SHBG),
38% bound to albumin and about 2% is free. As with hormones it is the free fraction that is
active. The free fraction of testosterone and the binding globulin are measured in various
diseases.
Basis of some of the disorders of sex differentiation and development: Sex is determined at
conception and reinforced at puberty. Sex differentiation is controlled by various genes
located on sex chromosomes. Male and female embryos have an inherent tendency to
feminize unless there is active interference from mascularizing factors.
Males
To understand the basis of some of the disorders of sex differentiation and development it
important to know testosterone synthesis in Leydig cells. Testosterone is a C19 steroid and
48
synthesis of steroids is basically similar in various organs, serve for presence or absence of
particular enzymes. That being the case, there are diseases in which an enzyme in the
synthetic pathway is absent or the activity is reduced (re-call monogenic diseases). Some of
the metabolites and enzymes in the pathway can be measured in the investigation of these
disorders.
Receptor defects: A number of diseases also arise from androgen receptor and post receptor
defects of DHT and testosterone. Such diseases result in testicular feminisation syndrome due
to lack of response to androgens. DHT and testosterone as well as hormones of hypothalamicpituitary axis are therefore elevated in such patients. Leydig cell hypoplasia: can occur as a
result of the cells failing to respond to βhCG and LH due to receptor defect. Testosterone level,
which is critical to male sexual differentiation of Wolffian ducts and external genitalia are low
thus affecting development and function.
Monogenic diseases: Besides CAH, there are a number of monogenic diseases or inborn errors
of metabolism of testosterone synthesis from cholesterol involving the different enzymatic
steps. Since Y chromosome is present, the patients have testes and mullerian structure
regression. The diseases may result in various ambiguous genitalia e.g. partially developed
wolffian ducts and the external genitalia can also be female. Some of these diseases only affect
testosterone synthesis while others affect synthesis of glucocorticoids and mineralocorticoids.
5-alpha reductase deficiency (monogenic): There can be defects in conversion of testosterone
to DHT due to 5-alpha reductase deficiency, which reduces testosterone to DHT that is more
potent than testosterone. 5-alpha reductase deficiency results in less developed prostate and
phallus and depending on severity, the external genitalia may be female. Since these are XY
individuals, there is mullerian ducts regression and wolffian duct development. Females with
5-alpha reductase deficiency are not affected.
Evaluation of male gonadal function
Investigations of infertility in males involve semen analysis as first line investigation and this
is followed by assessment of hypothalamic-anterior pituitary-testicular axis (See Figure 10.2
of text).
 hypothalamic-pituitary-testicular axis
 plasma testosterone (and/or metabolite) levels;
 plasma FSH and LH Levels
To re-cap
Testes have 2 major functions: sex steroid hormone (androgen) synthesis and secretion as
well as gametogenesis. Although sex steroids are produced in various parts of the body, the
most significant production organs in males and females are gonads and adrenal cortex.
Androgen synthesis occurs in Leydig cells and spermatogenesis in seminiferous tubules.
These 2 processes are controlled by LH and FSH, which are secreted by anterior pituitary
gland. These hormones are controlled by luteinising hormone releasing hormone (LHRH)
also called GnRH from hypothalamus.
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Steroidogenesis occurs in Leydig cells and is under the control of the HypothalamusPituitary-Leydig axis. Anabolic steroids suppress LH secretion by negative feedback
resulting in reversible testicular atrophy.
LHRH/GnRH: The hypothalamus secretes GnRH, a decapeptide that stimulates anterior
pituitary gland gonadotrophs to release LH and FSH.
FSH and LH: Secretion of GnRH is episodic and pulsatile, hence, episodic and pulsatile
secretion of LH and FSH (70-100 minutes). Since FSH has a longer ½ life, the peaks are not
obvious.
LH is a glycoprotein made up of 2 subunits, which are  and . Note that thyroid stimulating
hormone (TSH), FSH and hCG are similar to LH in that these 4 hormones have 2 subunits,
which are  and . The  subunits are very similar with respect to amino acid sequences but
differ in CHO components while the major differences lie in  subunits. LH and hCG both
stimulate testosterone synthesis and secretion by Leydig cells. Placental hCG stimulates
foetal testes to secrete testosterone prior to secretion of LH by foetal pituitary gland. The
major function of LH is stimulation of testosterone synthesis and secretion by Leydig cells.
The target for FSH in testis is Sertoli cells.
Testosterone: The major site for testosterone synthesis are Leydig cells and testosterone is
the major androgen synthesized in these cells. Testosterone levels rise to about ½ adult levels
in 1st 6 months after birth then fall remaining low until puberty. Plasma testosterone level is
constant in adulthood and declines by about 30% after the age of 70 years.
Testosterone synthesis is dependent on LH. Testosterone inhibits LH secretion via negative
feedback. Testosterone is secreted in a diurnal manner with highest level in early morning
and lowest level in early evening.
Prolactin is required for testosterone synthesis and it increases’ tissues sensitivity to
androgens. Having said that, please note that high level of prolactin inhibit GnRH release.
Epitestosterone is of interest in Sports Medicine. This testosterone metabolite with no known
biological significance is secreted from testes in roughly equimolar amounts with
testosterone. If exogenous testosterone is administered, the ratio of testosterone to
epitestosterone shifts upwards from the normal 1:1. A ratio of 6:1 is suggestive of androgen
abuse.
Other androgens: In addition to testosterone, testes secrete androstenedione,
dehydroepiandrostenedione (DHEA), progesterone and 17-hydroxyprogesterone in small
amounts. Please note that androstenedione, progesterone and 17-hydroxyprogesterone are
precursors of testosterone. The adrenal cortex is also a source of androstenedione and DHEA
in male and females.
Transport & metabolism of testosterone
Leydig cell hormones in blood are bound to plasma proteins. Testosterone is bound to
testosterone binding globulin (TeBG) also called gonadal steroid binding globulin (GBG).
TeBG also binds oestradiol 17 and plasma testosterone is about: 2% free, 60% bound to
TeBG and 38% bound to albumin and other proteins. In addition to daily variations,
testosterone level varies in an 8-30 day cycle with ranges 9-28% of mean values.
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The higher potency of DHT is due to its higher affinity for androgen receptors than
testosterone. DHT is important in differentiation of genitalia. 5 reductase deficiency results
in less developed prostate and phallus. Such individuals have normal testosterone levels but
reduced DHT and 5 metabolites. Females with 5 reductase deficiency are normal.
Oestrone and oestradiol 17 (oestrogens) in males are synthesized by Sertoli and Leydig
cells. The major portions of oestradiol 17 and oestrone however come from peripheral
conversion of testosterone and androstenedione, respectively.
The importance of oestrogens in males is not very clear, however, levels of oestrogens in
males are almost similar to those in females. Testosterone level is more in males than
females, hence the high ratio of testosterone to oestradiol in males prevents feminisation
rather than absolute levels.
Gynecomastia which is growth in breast in men is associated with increased
oestrogen/androgen ratio.
Functions of androgens (re-cap physiology)
Androgens including testosterone:
 Stimulate formation of male phenotype during sexual differentiation i.e. Wolffian
duct development
 Increase size of external genitalia. The penis and scrotum are increased in size.
Accessory organs such as seminal vesicles enlarge.
 Stimulate sexual maturation during at puberty such as induction of hair growth, pubic
and beard. Shoulders broaden and muscles enlarge.
 Increase linear growth and muscle mass.
 Lower pitch of voice due to enlargement of larynx and thickening of vocal cords.
 Induce aggression and boisterous behaviour and libido.
 Increased secretion of sebaceous glands, this is prevalent at puberty.
Such effects are ‘androgenic’ and related to growth and development of 2O sexuality.
‘Anabolic’ relates to effects on somatic cells such as liver, kidney, bone and muscle and
include stimulation of synthesis of proteins at the same time inhibiting protein breakdown.
The change to puberty in boys starts at 6-7 years and this is associated with increased
secretion of adrenal androgens, which are controlled by ACTH. Testes can hyper or hypofunction. Hyper-function is rare while hypogonadism can be hypothalamic, pituitary,
testicular or chromosomal.
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The severity and symptoms vary depending on whether hypogonadism develops before or
after puberty and if spermatogenesis or endocrine function is compromised. Loss of testicular
function is associated with infertility, high-pitched voice, lack of muscle development and
narrow shoulders. These patients are also tall and the condition is called eunuchoidism.
Female
Re-call reproductive development in males.
In females, lack of testosterone from ovary leads to regression of wolffian ducts and external
genitalia develop into clitoris, labiae majora and manora. Mullerian ducts develop into oviducts
due to absence of MIH. Sexual phenotype differentiation is completed in about 10-20 weeks in
females.
The major parts of the female reproductive system are vagina, uterus and fallopian tubes,
ovaries and breasts. The 2 ovaries weighing about 15g each contain germinal epithelium, made
of follicles in which an individual oocyte is surrounded by granulosa cells, which nourish and
protect the oocyte. Surrounding granulosa cells is a basement membrane, which in turn is
surrounded by theca cells. The latter synthesise and secrete androgens while granulosa cells
convert androgens into oestrogens.
Ovaries
Ovarian cells like testis are classified as germ or somatic:
Germ cells: The functions of ovaries are production of germ cell; ovum in follicles.
Oogogenesis is controlled by oestradiol 17.
Somatic cells: The somatic cells in the ovary (theca interna and granulosa) synthesize
androgens and oestrogens and is under anterior pituitary gland control.
There is interaction in the ovary in steroid synthesis between theca interna and granulosa cells.
This involves diffusion of androgens synthesised and secreted by theca interna across basal
lamina (found between theca and granulosa cells) into granulosa cells to be aromatized into
oestrogens by aromatase (Figure 10.4 in prescribed text). There is no blood barrier like in testis.
52
Figure: Oestrogen synthesis in females (Medbullets) This figure contains the same
information as in prescribed text
Female reproductive tract system development requires aromatase that converts androgens to
oestrogens. Full reproductive tract development in females takes 12-14 years and stops at
menopause. The development is continuous and involves GnRH from hypothalamus, LH and
FSH from anterior pituitary gland and oestrogens from ovaries.
Oestrogens and progesterone are the 2 major classes of hormones synthesized and secreted by
ovaries with the greatest biological activity. The oestrogens are oestradiol 17, which is the
major and most active oestrogen and oestrone and oestriol. Androgens (androstenedione and
testosterone) are also produced by ovaries. Note that aromatization of androgens to oestrogens
also takes place in peripheral circulation.
53
Transport
Ovarian steroids are derived from cholesterol and are carried in blood bound to proteins, these
protein bound sex hormones act as a reservoir in a similar way to how thyroid hormones
are bound by plasma proteins. About, 50% of oestradiol 17 is bound to TeBG, 48% to albumin
and 2% is free. Progesterone is transported; 50% bound to cortisol binding globulin, 48%
bound to albumin and 2% is free.
Function
Oestradiol and oestrone, maintain female sexual characteristics, prepare the uterus for
spermatozoal transport, increase vascular permeability, stimulate growth and activity of the
mammary glands and endometrium and prepare the endometrium for progesterone action.
Leading up to puberty, oestrogens promote: breast development, maturation of external
genitalia, female pattern of lipid distribution, there is extensive adipose tissue directed to the
hips, facilitates closure of epiphyseal plates at the end of linear growth, enlargement of hips
and pelvis to facilitate childbirth latter, inhibit bone resorption together with androgens. After
menopause, exogenous oestrogens are used to reinforce and maintain bone density, slow
development of dementia, suppress menopausal symptoms such as hot flashes and
depression.
Oestrogens are however associated with increased incidences on breast and endometrial
cancers, reduced milk secretion and thrombosis.
In some animals oestrogens stimulate oestrous behaviour.
Progesterone maintains the uterine endometrium and prepares it for implantation, maintains
the uterus during pregnancy and this involves inhibition of uterine contractions, stimulates
mammary gland growth (duct formation), but suppresses milk secretion.
Androgens: The ovaries produce testosterone, which is the 1O physiologically active
androgen in females and androstenedione. In females, androgens stimulate sex drive and
development of axillary hair at puberty.
Control of ovarian function
Hypothalamic-anterior pituitary gland-ovarian axis is controlled as follows: LH and FSH from
anterior pituitary gland are stimulated by GnRH. Secretion of LH is inhibited by oestradiol 17
β and progesterone by negative feedback. Oestradiol 17 β and progesterone also inhibit FSH
secretion. Furthermore, inhibin secreted by granulosa cells in developing follicles also
selectively inhibits FSH secretion. Therefore, basal FSH secretion is generally lower than LH.
LH surge, which triggers ovulation is positively-feedback controlled by neuroendocrine reflex.
LH surge is stimulated by high level of oestradiol 17 β that is secreted by Graffian or preovulatory follicle. In contrast to oestradiol 17 β, progesterone inhibits LH surge and this ensures
that oestrogen secretion in early pregnancy does not stimulate ovulation.
Menstrual cycle
A good understanding of the structural/biochemical changes (re-visit physiology notes) that
occur during the menstrual cycle is essential to grasp the details of the endocrine control of
54
reproduction in females. Understanding the menstrual cycle in terms of changes in anatomy
and the hormones secreted and their levels are pre-requisites to interpretation of hormonal
investigations in infertility and in understanding how some of the contraceptives work.
Understanding of hormone levels at each phase in the menstrual cycle is also important in
fertility treatment. The cycle is designed to produce oocytes for fertilisation.
Hormonal control of the menstrual cycle involves the hypothalamus-anterior pituitary glandovarian axis. The levels of oestrogens and progesterone, as well as the trophic hormones FSH
and LH follow a defined cyclical pattern during the female menstrual cycle, which is shown
in Figure 10.5 in the prescribed text. This pattern is the end product of a complex control
system involving the hypothalamus-anterior pituitary-ovary axis.
Figure: Menstrual cycle (Angea.com.au)
Note the following regarding the hypothalamus-anterior pituitary gland-ovary axis:
 GnRH a decapeptide produced by the hypothalamus stimulates the release of both
FSH and LH from the anterior pituitary gland;
 although FSH and LH are the major hormones affecting ovarian function, the pituitary
hormone prolactin also affects the corpus luteum. The major action of prolactin is on
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mammary gland tissue in initiating lactation. However, in some species prolactin is an
essential component of the luteotrophic complex (i.e. it is essential for the
maintenance of the corpus luteum). In women, prolactin may be important in
regulating follicular steroidogenesis and in the development of LH receptors thus
controlling progesterone secretion;
 unlike other hormone systems, prolactin release is normally inhibited by the
hypothalamus. The neurotransmitter dopamine released by the hypothalamus has the
major effect of inhibiting prolactin release by the anterior pituitary. Prolactin release
is also stimulated by a range of hypothalamic releasing factors including vasoactive
intestinal peptide (VIP) and thyrotropin releasing hormone (TRH); however the roles
of VIP and TRH are yet to be fully elucidated and normally dopamine appears to
override, keeping prolactin levels low. During suckling, dopamine release is inhibited
causing increased prolactin levels;
 prolactin inhibits the release of gonadotropin releasing hormone (GnRH) from the
hypothalamus. High prolactin levels inhibit the mid-cycle surge LH (and FSH) and
thus ovulation is suppressed. This effect has been used to explain the relative
infertility of lactating women (not to be relied on as a contraceptive !!!!!!).
Infertility and reproductive function aberrations
There are many known causes and equally there are also many unknown causes of infertility.
Infertility can occur due to dysfunction of any part in hypothalamic-pituitary-ovarian axis.
Oligomenorrhoea (infrequent menstruation), polymenorrhoea (short menstrual cycle) and
amenorrhoea (absence of menstruation) an be caused by abnormalities in the hypothalamicanterior pituitary gland-ovarian axis.
A common cause of infertility is amenorrheoa, which can be due to hyper or hyposecretion of
hormones in this axis. In normal women, during reproductive years, pregnancy is the most
common cause of amenorrheoa. Amenorrheoa can also be caused by prolactin-secreting
tumour of the pituitary gland, extremes in body weight and prolonged exercise in athletes.
One disease that alters ovarian structure resulting in altered function is polycystic ovarian
syndrome (PCO), which results in anovulatory (oocyte is not released) cycle. PCO can be
triggered by insulin resistance; hence, it is sometimes associated with weight gain. High
androgen levels are responsible for hirsutism, anovulation, infertility and uterine bleeding.
Anovulatory cycles can also be due to endocrinopathies such as hyperprolactaemia or
hypothyroidism (TRH stimulates prolactin secretion). Note that hyperprolactaemia inhibits LH
and FSH release. The incidences of breast and endometrial cancers are high in individuals with
anovulatory cycles.
Congenital adrenal hyperplasia (CAH): There are various types of CAH involving the
various enzymes in steroid synthesis. In all the types, there is impaired cortisol synthesis and
hyperplasia of adrenal cortex due to hypersecretion of adrenocorticotrophic hormone
(ACTH). Most types of CAH are predominantly virilising and involve prenatal
masculinisation of female foetus as a result of overproduction of adrenal androgens and
androgen precursors. This affects fertility later.
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Exogenous testosterone and progesteronal agents: Masculinisation of external genitalia in
females is sometimes seen following maternal injection of testosterone or progesteronal agents,
which are androgenic. The latter are taken in females with habitual or threatened abortions.
Endocrine evaluation of female ovarian function
Ovarian dysfunction can result in:
1. oligomenorrhhoea or amenorrhoea;
2. infertility or subfertility;
3. hirsutism and virility.
Ovarian dysfunction can result from either anatomical or endocrine abnormality; we shall
only consider endocrine measurements.
Just as with males we need to consider the hypothalamic-pituitary-ovarian axis
Ovarian hormone measurements
Two groups of hormones are commonly measured:
 plasma levels of oestrogens, especially oestradiol-17β: Measurements are usually
performed at three stages of the menstrual cycle-early follicular, mid-cycle (at
ovulation) and luteal. In amenorrhoea, there is no increase in plasma levels at midcycle. Whilst such measurements tell us that pathology exists, they do not pinpoint the
origin of the pathology;
 plasma progesterone: Measurements are performed at the early follicular and at the
luteal stages of the menstrual cycle. When ovulation has not occurred, there is no
increase in plasma levels at the luteal stage; once again the origin of pathology is not
identified.
Anterior pituitary hormone measurements
These measurements fall into two categories:
 plasma prolactin levels: this measurement is the primary measurement in cases of
amenorrhoea; the clinical significance of prolactin is described in the textbook.
Hyperprolactinaemia and resultant loss of ovulation are the most common cause of
female infertility. This is often caused by physiological or pharmacological causes
such as lactation, stress or the action of drugs. The most common pathological cause
is pituitary tumours;
 plasma FSH and LH levels: when plasma prolactin levels are normal, measurements
of plasma FSH and LH levels (especially the appearance of their mid-cycle surges)
are valuable in ascertaining whether the origin of pathology is at the level of anterior
pituitary or the ovary.
Note that measurements of plasma FSH, LH and oestradiol are commonly performed to
ascertain the exact time of ovulation for in vitro fertilisation procedures.
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Other measurements
Two other measurements are performed:
 plasma testosterone and dihydroepiandrosterone (DHAS) levels: these are
important in the evaluation of female hirsutism and virilism;
 the gonadotrophin releasing hormone (Gn-RH) stimulation test: this is used in
investigating e.g. the cause of delayed puberty.
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Topic 6 Endocrinology of pregnancy and other biochemical
changes in pregnancy
Rae P., Crane M., & Pattenden R. (2018). Clinical Biochemistry:
Lecture notes (10th ed.), Chapters 10 & 11. Wiley-Blackwell.
Assumed previous knowledge
Human Anatomy and Physiology/Physiological Sciences
 The physiology of fertilisation and pregnancy
Learning outcomes
 describe physiological events associated with pregnancy;
 describe the structure and significance of human chorionic gonadotropin (hCG);
 evaluate foeto-placental hormones and their usefulness;
 evaluate other biochemical changes associated with pregnancy and the investigations;
 describe gestational diabetes mellitus and pre-eclampsia.
Clinical biochemistry of pregnancy and parturition
a. Pregnancy is associated with various biochemical/metabolic, anatomical,
physiological and psychological changes.
b. Pregnancy is associated with stress and increased workload on various systems. This
can unmask latent diseases such as diabetes mellitus and heart diseases.
c. Pregnancy is associated with immune suppression, hence the mother is susceptible to
infection during pregnancy. Such suppression of immunity can be beneficial in some
autoimmune diseases.
d. Some drugs damage the foetus hence it is advisable to include or to exclude
pregnancy when prescribing to a female of reproductive age.
This discussion considers some of the clinical biochemistry aspects associated with
pregnancy so that we understand the reasons for some of the clinical biochemical
measurements during pregnancy. Measurements of hormones and other metabolites are
important to confirm pregnancy and to monitor pregnancy.
Fertilisation involves fusion of plasma membranes of oocyte and spermatozoa allowing the
spermatozoa to enter the cytoplasm of oocyte. The zygote then travels in the oviduct to the
uterus and during the journey it divides. By the time it reaches the uterus, which takes about 3
days, it is formed into a ball of cells called morula. At the same time, the uterus is prepared to
support the foetus. The endometrium is fully differentiated following preovulatory oestrogen
priming and postovulatory exposure to progesterone. The morula develops into blastocyst,
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which is hollow and made up of trophoblast cells, which are the outer surface and inner cell
mass that develops into the foetus. Trophoblastic cells initiate implantation onto the wall of
the uterus – progesterone is an absolute requirement for implantation. Trophoblast and
endometrium develop into placenta.
During implantation, trophoblastic cells move deep into the endometrium and fuse to form a
syncytium called syncytiotrophoblast. Implantation takes place about 7 days from the time of
fertilisation and at about two days, the trophoblastic layer of the blastocyst starts to synthesise
and secrete hCG, hence the presence of hCG in urine tests for pregnancy. The placenta serves
to exchange gases and nutrients between the mother and foetus and placental hormonal
secretion maintains pregnancy and it is also an endocrine organ that secretes two peptides;
hCG and chorionic somatomammotrophin (hCS), also called placental lactogen and two
steroids: oestrogen and progesterone, to maintain the foetus. The role of ovarian oestrogen
and progesterone is limited to early pregnancy, after which the placenta takes over. LH and
FSH are therefore low due to negative feedback and this ensures ovarian quiescence in
pregnancy.
Oestrogens: Pregnancy is associated with an elevated level of oestrogen secretion and after
the 4th week nearly all the oestrogens come from the placenta. The placenta however, cannot
convert C21 steroids to C19 (cannot directly synthesise oestrogens from pregnenolone),
although it can aromatise C19 steroids. The placenta depends on circulating C19 steroid
precursors for oestrogen synthesis. The major precursor for placental oestradiol 17β is DHEA
sulphate from the foetal adrenal gland and since the precursor of placental steroids comes
from foetus, hence, foetal-placental unit. In non-pregnancy, oestradiol 17β and oestrone are
the major products of follicular granulose cells, while in pregnancy, the major oestrogen is
oestriol. In non-pregnancy, the ratios of urinary oestriol:oestrone:oestradiol 17β are around
3:2:1, while in late pregnancy it is about 30:2:1. Oestriol is formed in placenta from foetal
adrenal C19 steroids. Oestrogens are therefore measured in pregnancy.
Progesterone: Progesterone peaks after 3-4 weeks of fertilisation, gradually declining during
gestation and rises again towards parturition. In the 1st trimester, progesterone comes from
corpus luteum, thereafter the placenta assumes the responsibility of progesterone secretion.
During the latter weeks of pregnancy, the placenta secretes large quantities of progesterone,
which is synthesised largely from maternal cholesterol.
Elevated level of progesterone is required throughout pregnancy, because after placenta
develops and the foetus begins to grow, progesterone inhibits myometrium contraction,
which can expel the foetus. Progesterone maintains endometrium and therefore sustains
pregnancy. Elevated progesterone concentration protects against premature labour. This is
because progesterone inhibits prostaglandin synthesis that induces uterine contractions. The
rise in progesterone towards parturition is associated with mammary gland development.
High levels of progesterone in pregnancy stimulates cervical secretions to become gelatinous,
which forms a mucous plug that obstructs the cervical canal. This plug prevents bacteria and
pathogens from entering the uterus which protects the embryo from infection. Progesterone
suppresses the mother’s immunity so the foetus is not recognised as foreign.
hCG: When implantation occurs, progesterone secretion by corpus luteum of menstruation is
maximal, but in the absence of a signal from the embryo, luteolysis occurs causing
menstruation and loss of the implanted embryo. To prevent this, one of the first events
following implantation is secretion of hCG, at about 1-2 days following implantation. This
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hormone is secreted by syncytiotrophoblasts and increases rapidly in the first few weeks of
pregnancy; maximum level is attained at about 10 weeks of gestation, after which the level
declines slowly. The hCG level is also increased in multiple pregnancy, choriocarcinoma
(cancer of placenta) and hydatiform mole (molar pregnancy). Measurement of urinary hCG is
a commonly used pregnancy test. Abnormal levels are seen in some chromosomal diseases.
Human placental lactogen (hPL) or human chorionic somatomammotrophin (hCS): This
polypeptide hormone is secreted by syncytiotrophoblast and is structurally similar to GH. The
concentration of hPL increases throughout pregnancy and is involved in development of
maternal breasts. The secretion commences on nidation (implantation of fertilised ovum in
uterus) and measurement of this hormone is used to assess placental function. Maternal hPL
parallels placental mass and it has lactogenic and somatotrophic (growth promoting)
properties. Its’ main function is to antagonise insulin (conferring insulin resistance) and is
responsible to a large extent for gestational diabetes mellitus in pregnancy. This anti-insulin
action serves to shunt glucose to the foetus. Glucose is routinely monitored in pregnancy for
fear of possible development of diabetes mellitus. hPL acts on the mammary gland to induce
enzymes for milk synthesis. Since placental lactogen is formed in trophoblasts, measurement
of this hormone assesses placental function and indirectly foetal viability.
Other hormones
Pregnancy is associated with changes in levels of hormones and hormone binding proteins. It
is important therefore to interpret endocrine data in pregnancy with care. Equally, hormonal
measurements are part of assessment and management of patients with endocrine diseases
such as thyroid and adrenal diseases.
Other biochemical changes: There are massive endocrine changes in pregnancy. Blood loss
is enormous and during normal vaginal delivery can be up 500 mL, while during caesarean
section, the loss can be as much as 1 000 mL. Blood volume increases in pregnancy but in
spite of this increase, blood pressure is low by comparison with non-pregnancy, due to
maternal vasculature loss of responsiveness to pressor effects (mediated by prostaglandins).
Failure in this adaptation accounts for some cases of pregnancy-induced hypertension.
During the first half of pregnancy, the mother is an anabolic phase. Protein synthesis and
glycogenesis in the liver and muscle are increased to facilitate growth and development of
breasts and the uterus as well as to allow the mother to store substrate for the demands of the
last trimester. In the second half of pregnancy, maternal metabolism is catabolic; the stored
substrate is consumed by the growing foetus, hence fasting hypoglycaemia is common. The
mother becomes insulin resistant to make glucose available to the growing foetus. The
transformation of anabolism to catabolism is due to hPL, cortisol, progesterone and
oestrogens. Elevated hPL levels increase lipolysis and this increases free fatty acids for
glucose formation. Normally, the foetus metabolises glucose but in starvation ketones can
also be metabolized.
Pre-eclampsia: is a syndrome characterised by hypertension, proteinuria and oedema. This
condition occurs after about 20 weeks of gestation, but the aetiology is not clear, perhaps it is
associated with abnormal implantation. If it is not managed, pre-eclampsia leads to
eclampsia, which is characterised by seizures and in these patients, water and electrolyte
imbalance, impaired renal function, proteinuria, abnormal liver function and hyperuricaemia.
These are therefore useful measurements in this condition (refer to BMS207).
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Gestational diabetes mellitus (refer to BMS207): is observed in about 4% of pregnancies and
is due to increased counter-regulatory hormones, as well as insulin resistance in pregnancy.
There is also an increased risk of diabetic complications, such as diabetic ketoacidosis,
retinopathy, nephropathy, hypoglycaemia and infections are common in diabetics during
pregnancy. These patients are at increased risk of developing pre-eclampsia and eclampsia.
Infant hypoglycaemia, hypocalcaemia and hyperbilirubinaemia are common in babies of
diabetic mothers.
Bone metabolism and the associated ions: There is significant mobilization of calcium from
mother to foetus to build foetal bones especially in the 3rd trimester. This puts the mother at
risk from bone de-mineralisation resulting in e.g. osteoporosis and bone fragility. In patients at
risk, calcium, phosphate, magnesium and the associated vitamins i.e. vitamin D3 and
parathyroid hormone need monitoring. Vitamin D3 and parathyroid hormone levels are
normally increased during pregnancy and such physiological elevations need to be taken into
account when interpreting this hormone in child-bearing adults. Maternal and foetal kidneys
and the placenta have 1-α-hydroxylase activity. There is also increased GIT absorption of
calcium in the mother perhaps as a result in increased vitamin D3 levels. Hypoalbuminaemia,
which is seen in pregnancy can lower total calcium levels.
Other biochemical tests
Pregnancy is associated with changes in fluid volume, which has a dilution effect on some
metabolites. GFR can increase due to the vasodilatory effects of progesterone and such
factors affect sodium, urea and creatinine levels.
In BMS207 we talked about placental isoenzyme of ALP this means that total ALP increases
in pregnancy and this should be minded.
Pregnancy is associated with changes in metabolic demands of the mother, foetus and the
developing mammary system as the pregnancy progresses. Hormonal changes (oestrogens,
progesterone and insulin) in pregnancy contribute to alter proteins, CHO and lipid
metabolism. Re-call that steroid hormones are synthesized from cholesterol. Note: the
increases in lipids: triglycerides and cholesterol predispose to CVDs.
Iron, transferrin and ferritin are needed for RBC synthesis by mother and foetus. Maternal
iron levels and storage may decrease and this may call for iron supplementation in pregnancy.
Therefore, related investigations are carried out viz iron measurement of levels of iron as well
as iron transport and storage proteins.
There are a battery of tests that are informative collectively and these are carried out to
improve predictability of various diseases inclusive of Down’ syndrome, (trisomy 21),
Edward syndrome (trisomy 18), Patau’s syndrome (trisomy 13), spina bifida, abnormal
abdominal wall defects. With these tests the final prediction takes into account e.g. age,
smoking and drinking habits, diabetes, metabolic syndrome, ethnicity and data from other
diagnostic departments such as ultrasound.
Alpha foeto protein (also covered in BMS207): produced by foetal liver from week 6, with
highest levels in the 2nd trimester. Its function in humans is not clear but possibly
immunoregulatory. In neural tube defects, this protein is elevated in amniotic fluid and
maternal blood. It is also a tumour marker.
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Unconjugated oestriol: produced by foetal placenta unit and low levels are associated with
Down’s syndrome, Edward’s syndrome, neural tube defects, restricted foetal growth and
other chromosomal and congenital abnormalities. As mentioned, during pregnancy oestriol is
the dominant major oestrogen and increases throughout pregnancy. During pregnancy this
hormone possibly regulates uteroplacental blood flow and vascularization as well as ensuring
inactivity of uterus in pre-labour pregnancy. Unconjugated oestriol is also used to predict
labour onset and there is interest if it can predict pre-term labour risk.
Cell free DNA: free DNA from placenta and foetus in the mother’s blood is used to assess
the risk for chromosomal diseases such as Down’s syndrome and trisomy 18.
Inhibin A: produced by foetal placental unit throughout pregnancy. Its role is not clear but
inhibins and activins in adults regulate FSH in both sexes and in males, inihibin A also
regulate spermatogenesis. It is now considered as a better marker than hCG since it has a
short half-life. It is useful in prediction of miscarriages, Down’s syndrome, pre-eclampsia,
among others.
Pregnancy associated plasma protein A (PAPP A): produced by foetal placental unit,
regulates insulin like growth factor that is vital in foetal development as well as protecting the
foetus from maternal immune system. The levels increase with gestational age and decrease
after delivery. Low levels are associated with increased risk of adverse pregnancy outcomes,
chromosomal anomalies, pre-eclampsia, miscarriages and early birth. High levels also appear
to be associated with risk of chromosomal anomalies.
Common battery of tests
Triple test (normally in 1st trimester): AFP (foetal liver), hCG (placenta) and unconjugated
oestriol (foetal placenta)
Quadruple test (normally in 2nd trimester): AFP, hCG, unconjugated oestriol and foetal liver)
and inhibin A (placenta). PAPP A can also be part of the quadruple test.
Parturition (not examinable)
Parturition involves expulsion of the foetus and placenta from the mother’s reproductive
tract. Parturition is complex and it involves interaction and integration of the maternal, foetal
and placental hormones.
Throughout pregnancy, weak Braxton-Hicks contractions are observed, which increase in
frequency and intensity just prior to the onset of labour. Please note that oestrogens stimulate,
while progesterone inhibits uterine contractions hence progesterone maintains pregnancy.
Whether it is the absolute level of oestrogens or progesterone or the increase in ratio of
oestrogens to progesterone, which initiate labour, is not clear. However, once the foetus has
reached a critical size, the muscles of the uterus are sufficiently stretched to stimulate
contraction.
Oxytocin: Oxytocin is important for parturition. Firstly, it maintains the expulsive phase and
secondly, oxytocin contracts the uterus in order to minimise blood loss after delivery.
Oxytocin is synthesised in the hypothalamus and the uterus-contracting activity of oxytocin
was one of its functions to be first identified. This property has also been utilised to induce
63
labour. In normal labour, the role of oxytocin in initiating labour is not clear, but there is no
doubt that oxytocin maintains labour. Reflex neural input from the stretched cervix and the
whole uterus to the hypothalamus stimulates oxytocin release. Oxytocin stimulates
contraction and the force and frequency are increased. After initiation of labour, maternal
oxytocin further increases and reflexes from the contracting uterus also feedback positively
on oxytocin release. Oxytocin also stimulates prostaglandin synthesis, which also increases
uterine smooth muscle contraction.
Oxytocin is also thought to reduce anxiety of exposure to the cry of the newborn and appears
to control maternal behaviour, stimulating maternal instincts and behaviour.
Relaxin: Relaxin is a polypeptide produced towards the end of pregnancy by corpus luteum
and relaxes the cervix, pelvic muscles and ligaments, inhibiting myometrial (uterine) activity.
The cervix that has been acting throughout pregnancy as a plug, preventing entry of
pathogens and toxins to the foetus, softens or ripens due to dissociation and reduction of its
collagen fibres, due to the effects of relaxin.
64
Topic 7 Endocrine dysregulation of calcium, phosphate and
magnesium metabolism
Rae P., Crane M., & Pattenden R. (2018). Clinical Biochemistry:
Lecture notes (10th ed.), Chapter 5. Wiley-Blackwell.
Assumed previous knowledge
Biochemistry
 Structure and synthesis of steroids and general structure of protein hormones
 Functions of hormones
Human Anatomy and Physiology/Physiological Sciences
 How endocrine organs work
 Location and structure of the parathyroid gland
 Structure of skeletal system
Learning outcomes
 describe the distribution of calcium, phosphate and magnesium in the body;
 describe the structure of bone;
 analyse the roles of GIT, bone and kidney in control of plasma calcium, phosphate
and magnesium levels;
 analyse the roles of hormones such as vitamin D3, parathyroid hormone (PTH) and
calcitonin in the control of plasma calcium, phosphate and magnesium levels;
 analyse the roles of pH, proteins and other factors in the control of plasma calcium,
phosphate and magnesium levels;
 describe synthesis and structure of PTH, calcitonin and vitamin D3;
 analyse disorders of calcium, phosphate and magnesium metabolism/levels;
 evaluate the tests for calcium, phosphate and magnesium disorders.
Introduction
The hormones, parathyroid hormone (PTH), calcitonin and calciferol with extracellular
calcium synergise and feedback upon each other to determine their own concentrations and
also the plasma levels of phosphate and magnesium. Calcium, phosphate and magnesium
regulate diverse functions from skeletal turnover to intracellular signalling. The control of
magnesium is not very clear but is linked in most cases directly/indirectly to calcium control.
65
Approximate distribution (grams) of calcium, magnesium and phosphate in the body
of a 70 Kg human adult () are % of each element.
Ca++
Mg++
HPO4-
Bone and teeth
1300 (99)
14 (54)
600 (86)
ECF
1 (0.1)
0.3 (1)
0.2 (0.03)
Cell
7 (1)
12 (46)
100 (14)
Approximate distribution (mM) of calcium, magnesium and phosphate in human adult
plasma () are % of each element (Figure 5.1 of text).
Ca++
Mg++
HPO4-
Protein bound
1.15 (40)
0.30 (30)
0.15 (13)
Free or ionized
1.10 (55)
0.50 (60)
0.60 (52)
Complexed
0.25 (5)
0.08 (10)
0.40 (35)
Please note that in Clinical Biochemistry ‘free’ and ‘ionised’ are sometimes used
interchangeably.
CALCIUM
Function
Calcium controls the major metabolic systems in the body.
a. Involved in some hormonal secretions.
b. Mediator of hormonal effects.
c. Neurotransmission: hypercalcaemia decreases neuromuscular excitability and
hypocalcaemia produces neuromuscular excitability, including muscle spasms.
d. Involved in cardiac and other muscular contractions.
e. Involved in blood clotting.
f. Activator of some enzymes.
g. Bone and teeth formation.
In addition, hypercalcaemia is associated with various cancers:
a. Cancer cells can invade bone, releasing calcium.
b. Some cancers e.g. of the breast, produce osteolytic compounds that resorb bone
releasing calcium and other minerals.
c. Some cancers e.g. lung, produce compounds with hormonal-like activity e.g. PTHlike activity.
d. Some cancers e.g. myeloma, are osteolytic. Furthermore, the hyperproteinaemia
associated with these cancers bind calcium, resulting in hyper-total-calcaemia.
e. Some cancers e.g. myeloma, lead to renal failure, which might elevate calcium due to
reduced GFR.
f. In the young, calcium deficiency leads to bone and teeth malformation, while in the
old, fractures are common due to weak bones and this leads to incapacitation. Cancers
such as myeloma that elevate plasma calcium are also common in the elderly; these
66
also lead to bone fractures. The social psychological, medical and financial
implications are enormous.
g. Increased plasma calcium concentration leads to renal failure due to calcium
deposition in the nephron, impairing glomerular and tubular functions.
h. Increased plasma calcium level is one of the disorders that leads to stone formation.
Renal stones can obstruct kidney function and obstruction of structures e.g. the ureter
is associated with infection leading to renal failure.
i. Disorders such as acidosis stimulate osteoclastic activity, increasing plasma calcium
concentration.
j. Disorders that lower plasma albumin level decrease total plasma calcium
concentration and vice versa.
Plasma calcium
Reference plasma range of calcium is 2.25-2.60 mmol/L and variation is controlled +10%
and equilibrium exists between ionized and protein bound. The ionized calcium is the
physiologically active fraction that directly affects properties such as neuromuscular
excitability and the release of PTH from parathyroid glands. The inactive protein-bound
calcium is attached mainly to albumin, with a small amount carried by globulins and
lipoproteins. Magnesium competes for the calcium binding sites.
The distribution of calcium between various forms is affected by plasma pH. Increased pH
decreases free calcium, while decreased pH has the opposite effect. Control of plasma
calcium, phosphate and magnesium concentrations depends on interaction of three hormones:
PTH, vitamin D3 and calcitonin. These hormones exert their control in an integrated manner
through three main processes:
a. Balance between the rates of deposition and mobilisation in the bone.
b. Absorption in the gastrointestinal tract.
c. Urinary excretion.
Bone
The bone consists of collagen fibrils and other proteins secreted by osteoblasts. Calcium,
magnesium and phosphate are deposited on/within the matrix. There are two major types of
bone cells involved in calcium metabolism:
1. Osteoblasts: bone forming cells which synthesise and secrete collagen (organic
matrix); this becomes the matrix on which calcification occurs. Stress due to gravity
and mechanics stimulates osteoblastic activity.
2. Osteoclasts: bone resorption depends upon osteoclasts, and these cells secrete H+ that
dissolves hyroxyapatite.
The bone is under constant remodelling, with some 5 mmol of calcium deposited and
reabsorbed each day. This balance can be disturbed, for example, by malignant disease, when
excessive calcium may be released from bone into plasma.
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GIT uptake and loss
Some 20-40% of daily intake of calcium (approx. 25 mmol) is absorbed by the gut. This
absorption is under hormonal control, therefore the GIT regulates body calcium level.
The actual amount absorbed depends on:
a. Amount of ionised or free calcium available. Free calcium in the intestinal lumen is
reduced by dietary substances that form calcium complexes, e.g. phytates and
oxalates. Acid pH increases formation of ionised calcium, while alkalinity promotes
complex formation and decreases absorption.
b. Vitamin D3, which increases absorption.
c. Dietary sugars, lactose stimulates calcium absorption.
Calcium is secreted into the gut as a normal constituent of bile and intestinal fluids. Under
normal circumstances the GIT is not an important excretion route for calcium.
Kidney
The kidney filters about 250 mmol of calcium each day and 98% is re-absorbed by the
tubules. The major fraction of this filtered calcium is taken up by the proximal tubules.
Plasma albumin
Alteration in the level of circulating albumin changes the concentration of bound and total
calcium. However, the concentration of ionised calcium is kept within reference ranges by
various factors, including:
a. PTH
b. Renal clearance
c. Re-equilibration with protein and other complexes
A fall or rise in plasma albumin of 10 g/L is usually associated with a fall or rise in total
calcium of 0.20-0.25 mmol/L. Distribution between albumin and free fraction is pH
dependent, as mentioned.
Calcium precipitation
Transient low calcium can arise if calcium is rapidly removed from plasma.
Hyperphosphataemia can, for example, cause the solubility constant of calcium phosphate to
be exceeded and the complex to be deposited in soft tissues. Similarly, high fatty acid levels
resulting from the action of lipase released during acute pancreatitis will also precipitate
calcium as calcium, complexes.
Hormones and calcium metabolism
Parathyroid hormone
Produced by chief cells of the parathyroid gland, PTH is polypeptide of 84 amino acids and
MW 9 300. It is formed as pre-pro PTH, which is converted to pro-PTH, from which 60
68
amino acids are removed for secretion as PTH. In circulation, there is further cleavage,
mainly in the liver to form two fragments (C-terminal and N-terminal). Only the N-terminal
is biologically active but the C-terminal fragment has a longer plasma half-life than the Nterminal fragment (what is the significance?).
Concentration of plasma ionised calcium regulates PTH secretion by negative feedback.
Magnesium has similar effect (unimportant) except in marked abnormal amounts. There is
also negative feedback by vitamin D3.
For maximal PTH activity, vitamin D3 is required, probably as a co-factor. Magnesium is also
necessary for maximum PTH response.
PTH acts on bone and kidney and also has an effect on GIT.
a. Bone
Stimulates osteoclast activity and conversion of stem cells to osteoclasts and this increases
bone resorption; hence, high plasma calcium and phosphate levels. The PTH activity on bone
is in two phases:
 Rapid response: transfer of calcium from the bone canalicular fluid into the osteocytes
and out into ECF.
 Delayed response: stimulation of osteoclast synthesis and activity, resulting in
resorption of bone. In addition, lysosomal enzymes are also released. Enzymes, such
as collagenase hydrolyse collagen releasing hydroxyproline into ECF ultimately
excreted in urine. PTH also inhibits osteoblast synthesis.
The effect of PTH in regard to bone is to increase plasma calcium and phosphate levels.
2. Kidney
PTH increases tubular reabsorption of calcium and decreases that of phosphate in the
proximal tubule. PTH also inhibits mechanisms for reabsorption of sodium, excretion of H+
and regeneration of bicarbonate. Proximal tubular acidosis is a feature in primary
hyperparathyroidism.
PTH increases synthesis of 1,25(OH)2D by increasing activity of 1-alpha hydroxylase in the
kidney.
The effect of PTH in regards to kidney is to increase plasma calcium and decrease plasma
phosphate levels.
3. GIT
Effects of PTH on GIT is indirect, through its effect on 1,25(OH)2D.
The effect of PTH in regard to GIT is to increase plasma calcium and phosphate levels.
The overall effect in plasma is: high calcium and low phosphate levels.
69
Vitamin D3 or [1,25(OH)2D]
This is a steroid that undergoes modifications before it can exert biological activity. The
majority of the body’s vitamin D3 requirements is met by the conversion of 7-
dehydrocholesterol, a sterol component of the epidermal layer of skin to cholecalciferol by
U/V light. With prolonged absence of exposure to sunlight, diet provides the sole source of
Vitamin D3.
Cholecalciferol arising from the skin bound to Vitamin D binding protein (DBP), together
with dietary ergocalciferol and cholecalciferol, are taken to the liver where they undergo
enzymatic hydroxylation to give 25-hydroxycholecalciferol (25-OHD) – also called calcidiol.
This intermediate is further hydroxylated by an enzyme in the kidney cortex 1-α hydroxylase
to 1,25-dihydroxycholecalciferol [1,25(OH)2D] also called calcitriol, the physiological active
metabolite of Vitamin D. 1-α hydroxylase activity is increased by PTH, hypophosphataemia
and hypocalcaemia and is negatively inhibited by 1,25(OH)2D.
There is some evidence that during periods of high calcium requirements (growth, pregnancy,
lactation) the relevant hormones (i.e. growth hormone and prolactin) stimulate formation of
1,25(OH2)D. The placenta also possesses 1-α hydroxylase activity, accounting for the
increased levels of 1,25(OH)2D in pregnancy.
If there is adequate 1,25(OH)2D then there is conversation of 25-OHD, via a separate renal
enzyme system, to a relatively inactive metabolite, 24, 25(OH)2 D. Both 1,25 and
24,25(OH)2D inhibit PTH secretion.
Actions of 1,25(OH)2D
1. GIT
Increases absorption of dietary calcium, magnesium and phosphate by stimulating the
synthesis of calcium-binding proteins (CaBP), such as calmodulin. CaBPs are located within
the intestinal cells and facilitate uptake of calcium. The mechanism by which it promotes
magnesium and phosphate uptake is not clear, other than that it differs from that of calcium.
The effect of 1,25(OH)2D in regards to GIT is to increase plasma calcium and phosphate
levels.
2. Bone
Mobilises calcium and phosphate from bone, a process that requires PTH. 1,25(OH)2D
stimulates osteoblastic activity hence is important for normal bone mineralisation to occur.
PTH activity on bone is impaired in the absence of 1,25(OH)2D.
The effect of 1,25(OH)2D in regard to bone is to increase plasma calcium and phosphate
levels.
70
3. Kidney
1,25(OH)2D enhances calcium and phosphate retention by the kidney. High levels of
1,25(OH)2D inhibit 1-α hydroxylase activity and stimulate 24-hydroxylase activity to
synthesise 24,25(OH)2D.
The effect of 1,25(OH)2D with regard to kidneys is to increase plasma calcium and phosphate
levels.
4. Parathyroid glands
1,25(OH)2D inhibits PTH production and indirectly inhibits PTH by increasing plasma
calcium levels. PTH and 1,25(OH2)D reciprocally influence one another through control of
plasma calcium and phosphate levels.
The overall effect in plasma is: high calcium and phosphate levels.
Vitamin D3 deficiency results in reduced mineralisation of bone, leading to rickets in children
and osteomalacia in adults. The bones become soft and are easily fractured. In osteoporosis,
minerals and matrix are lost and bone density is reduced, often resulting in the thinning of
bones and fractures. Osteoporosis is common in the elderly, especially in females due to
reduced oestrogen level that promotes bone formation and inhibits bone resorption.
Calcitonin
A polypeptide hormone (32 amino acids and MW 3 000) released by the parafollicular cells
(c cells) of the thyroid gland mainly in response to a high plasma calcium level. High plasma
calcium level stimulates calcitonin release, whilst low calcium has an inhibitory effect. The
role of this hormone in normal calcium metabolism in man is still controversial, but it has
been shown to have the following effects:
1. Bone
Opposes bone resorption by limiting the number and activity of osteoclasts, thereby lowering
calcium efflux from bone to ECF. Skeletal preservation may be the major physiological
function of calcitonin. Removal of calcitonin (e.g. thyroidectomy) does not apparently disturb
much calcium homeostasis or plasma level of calcitonin. It is however used as a tumour for
calcitonin producing c-cells of thyroid gland. The role of calcitonin remains unclear.
2. Kidney
Although pharmacological doses increase renal clearance of calcium and phosphate, it is not
clear whether this is a physiological role of calcitonin. Calcitonin also decreases synthesis of
1,25(OH)2D.
3. GIT
Effect on GIT through decreasing 1,25(OH)2D ??
The overall effect in plasma is low calcium and phosphate levels.
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Thyroid hormones
Influence rate of removal of calcium from bone; about 20% of thyrotoxicosis patients present
with mild hypercalcaemia. Thyrotoxicosis is also in some cases associated with decrease in
bone mass. Hyperthyroidism is associated with faecal and urinary loss of calcium.
Steroids
Androgens and estrogens: have anabolic effects and therefore stimulate osteoblasts and
calcium retention. Endocrine changes occurring at menopause are associated with loss of
bone mass; osteoporosis.
Growth hormone
Increases 1,25(OH)2D synthesis, therefore indirectly increases plasma calcium and phosphate
levels.
PHOSPHATE
Plasma phosphate levels:
Adults
0.6-1.4 mmol/L
Children
1.3-2.8 mmol/L
The above levels are for fasting. There is often a fall in plasma phosphate level after a
carbohydrate meal (what does this imply in measurement of phosphate?).
Functions of phosphate
 Skeletal.
 Carbohydrate metabolism.
 Energy transduction.
 Acid-base balance.
 Constituent of membrane, nucleic acids, phospholipids, phosphoproteins.
Homeostasis
GIT
About 90% of daily dietary intake of 25-35 mmol is absorbed and uptake is stimulated by
1,25(OH)2D.
Kidney
Renal handling of phosphate similar to glucose. Phosphate is reabsorbed by an active carrier
mediated process with Tm-limited characteristics. The rate of reabsorption is increased by
1,25 (OH)2 D and GH and decreased by PTH.
72
Bone
Phosphate is mobilised from the bone through the action of PTH (see notes on calcium)
Other factors
Acid-base: Acidosis is sometimes associated with a slight hyperphosphataemia while
alkalosis causes phosphate to enter cells, with resultant hypophosphataemia
Plasma glucose: Diabetes mellitus and its treatment are associated with disturbances in
phosphate metabolism. Insulin promotes phosphate entry into cells.
MAGNESIUM
Fourth most abundant cation in the body and second most abundant intracellular cation.
Distribution
Of the magnesium in the cytosol, 50-90% is complexed to phosphate, citrate and other ions
such as ATP. All enzymes utilising ATP interact with magnesium to form MgATP. Plasma
magnesium is 0.7-1.1 mmol/L, of which 30% is bound to plasma proteins, 12% is complexed
to various ligands and 60% is ionised.
Function
a. Chelates with important intracellular ligands such as ATP.
b. Required for enzymatic function: >300 magnesium activated enzyme described.
c. Maintains levels of intracellular calcium ions by competing for calcium binding sites
and by stimulating calcium sequestration by sarcoplasmic reticulum.
d. Maintains permeability hence electrical properties of membranes. Reduced
extracellular magnesium increases membrane excitability in tissues, such as heart.
e. Is found in structural proteins, polyribosomes, nucleic acids, probably confers
stability.
f. Neuromuscular action.
g. Action potential conduction.
Magnesium is important in hypertension since is associated with lowering blood pressure.
GIT
About 30% of dietary intake is absorbed in the small intestine. Magnesium uptake is
decreased by high levels of phosphate and fatty acids. Steatorrhoea is associated with loss of
magnesium. PTH appears to inhibit magnesium uptake through its stimulation of calcium
uptake that inhibits magnesium uptake.
PTH → ↑ 1,25 (OH)2D3 → ↑ Ca++ uptake→ ↓ Mg++ uptake
73
Kidney
Approximately 95-97% of filtered magnesium is reabsorbed and PTH increases renal uptake
of magnesium.
Disorders of calcium, phosphate and magnesium levels
The disorders are linked since the ions are controlled largely by the same hormones. Here we
concentrate on calcium disorders
Hypercalcaemia
Hypercalcaemia is more common than hypocalcaemia and carries the risk of stone formation
and renal damage.
Increased intake/absorption
 Vitamin D3 excess is usually a complication in patients taking the vitamin as part of
medication.
 Sarcoidosis: Rare disease of unknown aetiology, in which hypercalcaemia is caused
by a high sensitivity to vitamin D3. The biochemical picture is similar to vitamin D3
excess. Pulmonary alveolar macrophages from patients with sarcoidosis have been
shown to produce 1,25(OH)2D. This uncontrolled production of calcitriol in part
explains hypercalcaemia in these disorders.
 Milk-alkali syndrome: increased calcium and alkali intake. This syndrome is
characterised by hypercalcaemia, alkalosis and renal failure. It is due to excessive
intake of milk (vitamin D3 and calcium) and alkali (NaHCO3, MgCO3), usually for
treatment of ulcers. NB: Hyperparathyroid hypercalcaemia is associated with high
incidences of pancreatitis and gastric or duodenal ulcers.
Increased plasma albumin level
Increased bone resorption
 Malignancy: some neoplasms that cause osteolytic bone are carcinomas of breast,
kidney and thyroid, myeloma, leukaemia and Hodgkin’s and this results in dissolution
of bone elevating calcium and phosphate concentration in blood. Bone dissolution in
carcinoma can also be caused by secretion of an osteolytic sterol or substances that
stimulates PTH. The mechanism of hypercalcaemia in myeloma is not clearly
understood. Increased plasma calcium level is not entirely related to bone dissolution
or hyperproteinaemia.
 Although not the commonest cause of hypercalcaemia, primary hyperparathyroidism
is probably the best known.
 Thyrotoxicosis.
Increased renal absorption
 Familial hypocalcuric hypercalcaemia
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Others
 Paget’s disease: Hypercalcaemia can be a complication in Paget’s disease (excessive
breakdown of bone and disorganized remodelling). This will most likely occur if the
patients are confined to bed.
 Endocrine: Hypercalcaemia can be associated with thyrotoxicosis and
phaeochromocytoma.
Symptoms of hypercalcaemia include: muscular weakness, fatigue, headache, vomiting,
constipation, loss of appetite, irritable personality, polyuria, polydipsia; examination of eyes
might show corneal calcification, cardiovascular diseases.
Hypocalcaemia
Decreased intake
 Vitamin D deficiency
 Poor nutrition
 Malabsorption
Renal disease
 Reduced functional nephrons
 Decreased sensitivity to vitamin D3 and PTH
 Decreased conversion to 1,25(OH)2D
 Fanconi’s: defects in renal reabsorption can also be associated with defects in GIT
reabsorption.
Decreased plasma albumin
 Malnutrition
 Liver cirrhosis
 Nephrotic syndrome
 Protein-losing enteropathy
Decreased flux from bone
 PTH deficiency
 Resistance to PTH action e.g. in uraemia, magnesium deficiency
Pancreatitis
 Hypocalcaemia is secondary to malabsorption. Failure to absorb lipids in the GIT
results in formation of calcium soaps, which are not absorbed. Absorption of vitamin
D3 is also reduced.
75
Hyperphosphataemia
 Calcium absorption is reduced due to formation of calcium phosphate
Neonatal hypocalcaemia
 Poor feeding
 Immature vitamin D3 metabolism
 Parathyroid gland immaturity
Because of the role of calcium in neuromuscular transmission, low plasma calcium is
associated with tetany. Other symptoms include bone pain, convulsions.
Investigations
Plasma albumin and calcium
The concentration of total calcium is directly affected by that of albumin and does not
necessarily reflect the level of plasma free calcium. Free calcium level is the assay of choice,
but the methods are technically difficult and expensive. The problem can be partly alleviated
by measuring calcium and albumin levels in blood. Assuming plasma albumin level is 40 g/L,
there is a 0.02 mmol/L change in plasma calcium level for each 1 g/L in
albumin. Measurement of ionised/free plasma calcium is the most ideal.
Plasma phosphate
Plasma phosphate level is high in some cases of hypoparathyroidism and low in
hyperparathyroidism. Increased plasma calcium levels may result in renal failure; therefore
might also increase plasma phosphate as a tertiary response. Note: phosphate is high
intracellular, therefore haemolysed specimens affect analytical results.
Plasma ALP
ALP isoenzyme is secreted by osteoblasts, but the precise role of ALP in bone mineralisation
is not certain. However, it is elevated in growing children and adults over the age of 50 years.
High plasma ALP activity in conjunction with high plasma calcium level suggests high
osteoblastic activity, as would be expected in primary hyperparathyroidism and malignancy
of bones. ALP activity could also elevate due to liver involvement as a result of metastases
from a malignancy.
Please note that there are numerous other causes of high ALP activity, some which are
physiological, e.g. pregnancy. Therefore, ALP isoenzyme analysis is useful.
High values of bone ALP are indicative of Paget’s disease, osteomalacia and bone cancer.
Renal function
Renal dysfunction can result in low plasma calcium and high phosphate. High plasma
calcium level can result in renal dysfunction.
76
Plasma PTH
Plasma PTH can be technically difficult to measure since it has a short half-life and therefore
is unstable. In primary hyperparathyroidism and chronic renal failure plasma PTH level is
usually high.
Plasma calcitonin
Plasma vitamin D3 metabolites
1,25(OH)2D (calcitriol) has been used in investigation of vitamin resistance and intoxication.
Plasma HCO3-
PTH decreases H+ secretion and HCO3- regeneration, whereas hypercalcaemia due to other
causes is associated with this situation.
Urine calcium
High plasma calcium from any cause elevates urine calcium excretion. Familial hypocalcuric
hypercalcaemia is associated with a low renal calcium excretion rate hence measurement of
urine calcium is useful in such cases.
Urine hydroxyproline
Plasma and urine levels of the two amino acid constituents of collagen, i.e. hydroxyproline
and hydroxylysine, are high when collagen turnover is high (increased bone dissolution).
There is need to monitor patent’s diet (no galatin) for this test.
Urine cAMP
At cellular level, PTH acts via cAMP, therefore excretion of cAMP is high if PTH is raised,
due to, e.g. hyperparathyroidism or ectopic production.
Stone analysis
Calculi forms when the concentration of certain substances in urine exceeds saturation point.
Calcium oxalate calculi are the most common type of calculi and they tend to occur:
 If urine volume falls to below 500 mL/day.
 Following an increase in urine calcium. Any cause of hypercalcaemia may precipitate
stone formation.
Although calcium stones are typically composed of oxalate, varying proportion of phosphate
may also be present.
77
END

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