Notes
Notes
Notes
- Dehydration: Loss of fluid in ICF and ECF due to increased urinary losses
Figure 3.Water volume expansion
Osmolality
Major electrolytes: Na+, K+, Ca2+, Mg2+, Cl-, HCOO3-, HPO43- and SO42-
Number of dissolved particles in body water is measured by osmolality
Dissolved particles exert osmotic pressure across semi-permeable cell membrane, causes
water to flow into compartments with high conc. of dissolved solutes relative to other
compartments (movement from low solute conc. to high solute conc.)
E.g.:
- If cell is placed in a solution with high concentration of solutes (hypertonic solution), water will
move out of the cell, causing it to shrink or undergo plasmolysis.
- If cell is placed in a solution with lower concentration of solute (hypotonic solution), water will
move into cell, causing it to swell or undergo cytolysis.
- In isotonic solution, both sides of the membrane have equal concentration, there is no net
movement of water.
Role of semi-permeable membranes is to move water freely.
Osmotic pressure must always be the same on both sides of cell membrane (Osmolality of
ECF and ICF are normally the same, which is to prevent osmotic imbalances that could affect
cell shape and function)
This is achieved by movement of water across the semi-permeable membrane to keep the
osmolality the same, even if this water movement causes the cells to shrink or expand in
volume.
Osmolality expressed as mmol solute per kilogram of solvent (usually water)
Plasma/serum osmolality can be measured directly by using osmometer.
Osmolarity is the number of osmoles of solute per litre of solution
Calculation of osmolarity, if the concentration of major solutes is known:
- Calculated osmolarity= 2 (Na+) + 2 (K+) + Glucose + Urea (mmol/L)
- Calculated osmolarity= 2 (Na+) + Glucose + Urea (mmol/L)
Osmolality of Regulation
75% of Na is exchangeable and is in ECF, the concentration is around 140mmol/L. (remaining 25%
of sodium is found in bone and other structure that not as readily exchangeable)
Regulation of Na is done through kidneys.
If Na concentration increases, water need to be consumed to maintain osmolality by diluting the
Na. This in turn increases fluid volume and blood pressure.
Urinary Na output is regulated by 2 hormones:
(i) Atrial natriuretic peptide (ANP)
(ii) Aldosterone
Aldosterone
Angiotensin II has several physiological effects such as it causes vasoconstriction of blood vessels,
leading to an increase in BP. It stimulates the release of aldosterone from adrenal glands and ADH
from hypothalamus, and it promotes thirst, encouraging water intake.
Aldosterone acts on the kidneys, promoting the reabsorption of sodium and water and the
excretion of potassium. This helps increase blood volume and BP.
ADH promotes water reabsorption in the kidneys, further contributing to an increase in blood
volume.
As BP and volume increase, the release of renin decreases, providing a negative feedback loop
that helps regulate the system.
Loss of Fluids
(i) ICF loss: cellular dysfunction, notably evident as lethargy, confusion, coma
(ii) ECF loss: circulatory collapse, renal shutdown and shock
Electrolytes replacement
Na+ is present in highest conc. and makes the largest contribution to total plasma osmolality.
K+ conc. is low in ECF but compared to ICF even minor are very important.
Common scenarios when electrolytes replacement is require:
- Dehydration
- Exercise and sweating
- Medical conditions
- Malnutrition or imbalanced diet
Sodium Metabolism- Hyponatraemia
Definition: Serum sodium concentration below reference interval of 135-145mmol/L
Caused by:
- Retention of water
Na+ is the main extracellular cation which function in maintaining of blood volume and BP by
osmotically regulating passive movement of water.
When there is a decrease in sodium (Na+) levels, water is also lost, leading to the appearance of
typical signs linked to the depletion of the extracellular fluid (ECF) compartment. (The loss of
water from ECF results in decrease ECF volume, contributes to signs and symptoms such as low
BP, increased heart rate and dizziness. The movement of water into cells affects the ICF volume
leading to cellular swelling.)
Sodium Loss
Water Retention
- In healthy state, AVP conc. fluctuates between 0-5pmol/L, due to changes in osmolality. (When
blood becomes more concentrated, AVP released to promote water retention and concentration
of urine)
A phenomenon that occurs when measured sodium concentration in the blood appears low, but
the actual concentration of sodium in plasma is normal.
This phenomenon is typically related to changes in water content of blood sample rather than
actual imbalance of sodium.
Hyponatraemia is sometimes reported with patients with severe hyperproteinaemia or
hyperlipidaemia.
The increased amounts of protein or lipoprotein occupy more of plasma volume than usual and
the water less. (When blood sample contains high levels of lipids and proteins, the water
component of the blood is reduced in proportion.)
Na+ and other electrolytes are distributed in water phase only; these patients have normal Na+
in their plasma water.
However, many analytical instruments measure the sodium concentration in the total plasma
volume. It takes no account of water fraction that occupies less of the total plasma than usual.
An artefactually low sodium result may be thus obtained in such circumstances.
Water Loss
Sodium Gain
Clinical Features
Mild hypernatremia
Na+ 150-170mmol/L
Pure water loss likely if clinical signs of dehydration are mild in relation to severity of the
hypernatremia. This is because pure water is loss is distributed evenly throughout all body
compartments (ECF and ICF); the Na+ is unchanged
Gross hypernatremia
An acid is any molecule that able to donate a H+ ion (The donation of H+ ions to the solution
increases the concentration of hydrogen ions in the solution)
A base is any molecule able to accept an H+ (The acceptance of H+ ions by a base lead to a
decrease in the concentration of free H+ ions in the solution. As H+ conc. decrease, the pH
solution increase, more alkaline)
The products formed by acceptance of a H+ is dependent upon the nature of the base.
If the base is an acid anion (bicarbonate), an dissociated acid (carbonic acid) will produced.
Normal pH
Buffer Systems
Buffer acts like sponge which mops up excess [H+] ions but they do not remove ions.
A buffer solution of the salt (bicarbonate) of a weak acid (carbonic acid) is able to combine with
hydrogen ions.
The weak acid mentioned is carbonic acid (H2CO3), and its salt is bicarbonate (HCO3-).
The bicarbonate ion (HCO3-) has the ability to combine with hydrogen ions (H+) in a
reversible reaction. HCO3−+H+⇌H2CO3
When excess hydrogen ions (acid) are added to the solution, the bicarbonate ions can
combine with them, neutralizing the excess acidity.
If there’s a shortage of hydrogen ions (base), the bicarbonate ions can release hydrogen ions
to neutralize the excess alkalinity.
Buffering is only a short term solution until the body actually excretes excess hydrogen ions via
the lungs or via the kidneys.
Henderson-Hasselbalch Equation
The bicarbonate buffer system is present in the plasma and in the RBCs, and is the first line
defence against changes in [H+].
The relationship between [H+], carbonic acid and its dissociation to CO2 can described by the
Henderson-Hasselbalch equation.
The equation states that pH equals to the sum of pK plus the logarithm of the ratio of
bicarbonate to carbonic acid.
The normal ratio of bicarbonate to carbonic acid is 20:1
Carbon dioxide reacts with water to form carbonic acid. Carbonic acid dissociates into
bicarbonates ions and protons. Bicarbonate ions acts as buffer, readily accepting protons to
neutralize excess acidity or donating protons to counteract alkalinity.
Respiratory Buffer System
Bicarbonate buffer system: After combining with a hydrogen ion to form carbonic acid, this can
then dissociate to carbon dioxide and water.
The carbon dioxide can then excreted via the lungs which ensures that the system does not reach
equilibrium and can continue to buffer more hydrogen ions.
By removing CO2, the equilibrium in the bicarbonate buffer system is shifted to the left
(dissociation of carbonic acid), favouring the production of more bicarbonate ions. This
prevents the system from reaching a state of equilibrium and allows it to continue buffering
additional hydrogen ions.
The ongoing removal of CO2 ensures that the bicarbonate buffer system remains dynamic
and can continue its role in buffering, maintaining the pH.
The lungs therefore represent the next line of defence against changes in [H+] after plasma
buffers can respond in minutes by increasing or decreasing ventilation.
When all the bicarbonate is used up, the system has no more buffering capacity.
This lead to a loss of buffering capacity, making the solution more vulnerable to rapid shifts in
acidity or alkalinity. The depletion of bicarbonate can result in disruptions in acid-base balance,
potentially causing acidosis or alkalosis.
Renal Buffer System
Renal Buffer System can respond within hours to days to an acid-base balance.
Any hydrogen ion which is derived from non-volatile acids and buffered must eventually be
excreted from the body by the kidneys.
Secretion of hydrogen ion into urine allows the bicarbonate to recovered by the kidney.
Regeneration of bicarbonate also takes place in the kidney.
Hydrogen ion excreted in the tubular fluid is buffered by phosphate and ammonia ions in
order to prevent the urine becoming too acid.
Reference Ranges (Arterial)
pH (7.32-7.38) α¿ ¿
(pH is proportional to the ratio of bicarbonate and pCO2)
Distribution
Calcium: Phosphate:
Skeleton- 99% Skeleton-90%
Muscle- 0.3% Intracellular-5%
Other tissues-0.7% Extracellular- Less than 0.03%
Dietary Sources:
Calcium: Phosphates:
- Milk and Milk products - Same as calcium
- Egg - Present in high amount in cereals and
- Fish pulses
- Vegetables
- Fruits (oranges)
- Nut
Function of Calcium
Muscle contraction
Formation of bone and teeth
Nerve transmission
Preservation of cell membrane integrity and permeability
Resting membrane potential of the cells
Release of certain hormones
Major structural elements in the vertebrate skeleton (bones and teeth) in the form of
calcium phosphate (Ca10(PO4)6(OH)2) known as hydroxyapatite
Maintenance of cell structure
Membrane rigidity, permeability and viscosity are partly dependent on local calcium
concentrations.
Functions of Phosphate
Formation of bones
Important component of teeth
Important constituent of cells
Forms energy rich bonds in ATP
Forms co-enzymes
Regulates blood and urinary pH
Forms organic molecules like DNA and RNA
Absorption of Calcium
Taken through dietary sources such as calcium phosphate, carbonate, tartrate and
oxalate.
Absorbed from GIT into blood and distributed to various parts of the body
Two mechanism of calcium absorption by gut mucosa:
(i) Simple Diffusion
(ii) Active transport process (involves energy and calcium pump)
While passing through kidney, large quantity of calcium is filtered in glomerulus.
From the filtrate, 98-99% of calcium is reabsorbed in renal tubules into blood and only
small quantity is excreted through urine.
In the bone, calcium may deposited or resorbed depending on the level of calcium in
plasma.
Vitamin D
Pregnancy and growth
PTH
Vitamin-D
Action on Intestines
Action on bone
In osteoblasts of bone, calcitriol stimulates Ca uptake for deposition as CaPO4.
Calcitriol facilitates the absorption of calcium and phosphate from the intestine, ensuring
a sufficient supply of these minerals for bone mineralization. Moreover, it stimulates the
activity of osteoclasts, cells responsible for breaking down bone tissue and releasing
calcium and phosphate into the bloodstream. Simultaneously, calcitriol inhibits the
secretion of PTH, a regulator of bone resorption. By suppressing PTH, calcitriol helps
prevent excessive bone breakdown. Additionally, calcitriol promotes the function of
osteoblast, cells involved in building new bone tissue. This dual in regulating both bone
resorption and formation contributes to maintenance of optimal bone mineral density.
Action on kidney
It is involved in minimizing the excretion of Ca and P through kidney by decreasing their
excretion and enhancing reabsorption.
Pregnancy and growth
Pregnancy
During later stage of pregnancy, greater amount of calcium absorption is seen
50% of this calcium is used for development of foetal skeleton and the rest is stored in
bones to act as a reserve for lactation
This is due to increased level of placental lactogen and oestrogen which stimulates
increased hydroxylation of Vit-D.
Growth
Vitamin D
(i) Cholecalciferol/ D3
(ii) Ergocalciferol/ D2
Called as hormone because it produced in the skin when exposed to sunlight
Vit-D has very little intrinsic biological activity
Vit-D itself is not an active substance, it must be first converted through a series of
reaction in the liver and kidneys to final active produce 1,25-DHCC (Calcitriol)
Daily requirement
Adult: 2.5mg
Lactating mother, pregnancy, adolescents, infants: 5mg
Dietary Sources
Actions of Vitamin D
Actions of PTH
Calcitonin (Thyrocalcitonin)
Action of Calcitonin
Phosphorous excretion
Clinical Importance
(i) Hypercalcaemia
Elevated serum calcium level: 12-15mg/dl (2.1mmol/L-2.7mmol/L)
Conditions leading to hypercalcaemia:
(a) Hyperthyroidism (influence bone resorption, altering calcium and vit-D metabolism)
(b) Acute osteoporosis
(c) Thyrotoxicosis
(d) Vit-D intoxication
Signs:
- Diminished reflexes
- Short QT interval on ECG
Symptoms:
- Polyuria
- Dehydration
- Confusion
- Depression
- Fatigue
- Nausea/ vomiting
- Anorexia
- Abdominal pain and renal calculi
(ii) Hypocalcaemia
Decreased level of calcium in blood (<4mg/dL)
Conditions leading to hypoglycaemia:
- Insufficient dietary calcium
- Hypoparathyroidism
- Insufficient vit-D
- Increased in calcitonin levels
Symptoms:
- Irritability
- Muscle cramps
- Depression
- Bronchospasm and seizures
Vitamin D deficiency
(i) Rickets
Occurs in children between 6 months to 2 years of age
Affects long bones
Lack of calcium causes failure of mineralization resulting into formation cartilaginous
form of bone
Most critical area that gets affected: Central endochondral ossification at epiphyseal
plates
(ii) Osteomalacia
Softening of bones caused by not having enough vit-D or by problems with
metabolism of this vitamin.
These softer bones have a normal amount of collagen that gives the bone its
structure, but they are lacking in calcium.
Occurs in adults
Flat bones affected
Softening and distortion of skeleton bone
Symptoms:
Fractures of brittle bones occur even after minor accident
Pain due to fractures of vertebrae which may radiate round the trunk, to the buttocks
or down the legs
Oral manifestation include alveolar bone loss resulting in ill-fitting dentures and
periodontitis.
Alveolar bone loss: The part of jawbone that surrounds and supports the teeth. Loss
of this bone can have significant consequences for dental health
Ill-fitting dentures: Dentures are artificial teeth that replace natural teeth. Alveolar
bone loss can result in changes to the jaw structure, making dentures fir poorly and
uncomfortably.
Periodontitis: Severe form of gum disease, involve inflammation and infection of
structure supporting teeth, including alveolar bone.
Prevention:
Physical activity
Androgen and Oestrogen
Increased calcium intake and strontium and sodium fluoride ingestion
Hyperparathyroidism
(i) Primary hyperparathyroidism
A primary abnormality of the parathyroid glands causes inappropriate, excess
PTH secretion
Caused mainly by an adenoma of parathyroid
Hyperphosphatemia
Effects of Hyperphosphatemia
Phosphate trapping
Respiratory insufficiency
Erythrocyte dysfunction
Nerve Dysfunction
Leukocyte Dysfunction
Metabolic acidosis
Treatment
Treatment
Hypophosphatasia
Basic disorder is a deficiency of the enzyme alkaline phosphatase in serum or tissues and
excretion of phosphoethanolamine in the urine.
Clinical features:
- Infantile: severe rickets, hypercalcemia, bone abnormalities and failure to thrive
- Childhood: Premature exfoliation of deciduous teeth, increased infection, growth
retardation, rachitic like deformities, pulmonary, GIT, and renal abnormalities
Oral manifestation:
- Loosening and premature loss of deciduous teeth, chiefly the incisors.
Tutorial Questions:
Strategy of investigation of hypercalcemia and hypocalcaemia?
Investigation of hypercalcemia
The reference interval of calcium ion is 2.1 to 2.7mmol/L. From total calcium test, it is
supposed to be hypercalcemia as the serum calcium ion concentration shows above
2.7mmol/L. Hypercalcemia is mainly caused by the abnormal PTH level. If PTH level is higher
than normal range, it is related to the clinical conditions like primary hyperthyroidism with
low phosphate and high calcium levels and familial hypocalciuric hypercalcemia shown as
decreased urinary calcium levels. Also, insufficient PTH level can be compensated by
excessive production of other hormones. PTH2-peptide is the principal cause of
hypercalcemia malignancy. The granulomatous disease like sarcoidosis might be induced
since the calcitriol concentration increased. Vitamin D intoxication generally results from
elevated calcifediol synthesis. There are other possible causes of leading to hypercalcemia
with PTH and vitamin D level is normal.
Chapter 4: Endocrinology
Hormone: Any substance in an organism that carries a “signal” to generate some sort of
alteration at the cellular level.
(i) Endocrine hormones:
- Act on cells distant from the site of their release
- Examples: Insulin, glucagon, epinephrine, steroid hormones
- Endocrine signals: Directed at distant cells through intermediacy of bloodstream
(iii) Autocrine
- Act on the same cell that release them
- Examples: Interleukin-2 (Stimulates T cell proliferation)
- Autocrine signals: Directed at the cell that produced them
(ii) Steroid
- Example: Aldosterone, Testosterone, Cortisol and oestrogen
Polypeptide Hormones:
Hydrophilic (Water-soluble)
Do not penetrate plasma membranes, so it must bind to receptors in plasma membranes
of target cell.
The extracellular signal of a hormone get transmitted into the cell by generating second
messenger (e.g.: cAMP)
Second messenger carry information from first messenger hormone into the cell.
The second messenger are often produced using common proteins associated with
plasma membrane called G-proteins.
Once activated, the G-protein dissociates into an alpha subunit and a beta-
gamma complex. The specific target of the activated subunit depends on the type
of G-protein involved. In the case of the stimulatory G-protein, GS, the alpha
subunit activates the enzyme Adenylyl cyclase. This enzyme, in turn, converts ATP
into cAMP, a crucial second messenger.
Hormone glucagon travel through the blood stream to liver and bind to G-protein
coupled receptor (GPCR). This initiates an increase in cAMP, which leads to generation of
pKA resulting increase the glucose level by two ways:
(1) Blocking glycogen synthesis
(2) Accelerate glycogen degradation
When the body requires an increase in blood glucose levels, hormones like epinephrine and
glucagon are released. These hormones activate a signalling pathway that involves the
production of cAMP, which then activates pKA. pKA plays a central role in cellular responses
and phosphorylates glycogen synthase kinase (GSK). When GSK is activated, phosphorylates
and inhibits glycogen synthase, causing it to transition from an active ‘a’ form to an inactive
‘b’ form. In this inhibited state, glycogen synthase is less effective in catalysing the addition
of glucose units to glycogen chains, thereby blocking the synthesis of glycogen.
Accelerate glycogen degradation
When the body requires an increase in blood glucose levels, hormones like epinephrine and
glucagon are released. These hormones activates a signalling cascade that involves the
conversion of ATP to cAMP, leading to activation of pKA. pKA in turns activates
phosphorylase kinase.
For proper cell function, the cell must be able to stop the G-protein signalling pathway
after it has accomplished its task.
To terminate the signal, cAMP is broken down using enzyme cAMP phosphodiesterase.
The catalytic subunits of pKA then re-associate with regulatory subunits.
To inactivate G-protein, alpha subunit is hydrolyze its bound GTP back into GDP using
GTPase activity.
Alpha units then re-associate with beta-gamma complex, and the G-protein is once again
back to inactive state.
Finally, the cell is ready to be stimulated by another hormone.
Ca++, IP3 and DAG as second messenger
To terminate this signal, calcium is re-sequestered in the endoplasmic reticulum and PIP2
is formed.
The alpha subunit of the G-protein hydrolysis its bound GTP into GDP, and the G-protein
reassociate.
This restores the resting state of the cell so that another hormone can initiate cellular
effects.
Steroid hormones exert their physiological effects through a multi-step process that involves
intricate interactions with target cells. Initially synthesized and released by endocrine glands,
such as the adrenal glands and gonads, these lipid-soluble hormones navigate through the
bloodstream, often bound to carrier proteins. Upon reaching target cells, steroid hormones
diffuse through cell membranes and bind to specific intracellular receptors located in the
cytoplasm or nucleus. This binding triggers a conformational change in the receptor, forming
a hormone-receptor complex. In some instances, this complex translocate into the cell
nucleus. Once in the nucleus, it will binds to specific DNA sequences known as hormone
response elements (HREs), initiating or enhancing the transcription of specific genes. This
transcriptional activation leads to the synthesis of messenger RNA, which upon existing the
nucleus, directs the synthesis of proteins or enzymes in the cytoplasm. The newly
synthesized proteins then mediate the physiological response associated with the steroid
hormone, contributing to the regulation of diverse processes such as metabolism,
development and immune responses.
Neurons in the hypothalamus secrete thyroid releasing hormone (TRH), which stimulates
anterior pituitary to secrete thyroid-stimulating hormone (TSH). TSH binds to receptors on
epithelial cells in the thyroid gland, stimulating synthesis and secretion of thyroid hormones,
which affect probably all cells in the body. When blood concentration of thyroid hormones
increases above a certain threshold, TRH-secreting neurons in the hypothalamus are
inhibited and stop secreting TRH.
Positive feedback control:
Occurs when the rate of a process increases as the concentration of the product
increases.
The stimulation of a baby sucking its mother’s breast leads to secretion of oxytocin into the mother’s
blood, which leads to milk being available to the baby via the breast. The mother’s production and
release of oxytocin ceases when the baby stops feeding.
The positive feedback mechanism involved in the secretion of breast milk is a vital process ensuring
the effective initiation and sustenance of lactation. Initiated by the suckling stimulus of an infant,
nerve signals are transmitted from the nipple to the hypothalamus in the brain. In response, the
hypothalamus signals the posterior pituitary gland to release oxytocin, a key hormone in milk
ejection. Oxytocin in turn acts on the smooth muscles surrounding the milk-producing glands (aveoli)
and ducts within the breast. The action induces the milk ejection reflex, leading to contraction of
muscles and the release of stored milk into the ducts. The process is characterized by positive
feedback, whereby the continued suckling of the infant perpetuates the releases of oxytocin. This
self-reinforcing loop ensure that the more the baby suckles, the more milk is ejected, facilitating
ongoing nutritional supply required for the infant’s well-being.
Chapter 12
Vitamin A function:
i. Normal synthesis of mucopolysaccharides and growth of epithelial tissue for growth and
development
ii. Maintenance of immune system
iii. Needed by the retina of the eye in form of retinal, which combines with protein opsin to
form rhodopsin
Vitamin A:
Vitamin A Deficiency:
Mild Deficiency:
Vitamin E (α,β,γ,δ-tocopherol)
Function:
Deficiency:
Excessive Intake
Vitamin K
Functions:
Deficiency:
1. Hypoprothrombinaemia
- Less Vit.K synthesizing microorganisms in the intestine
- Patients on long-term antibiotic therapy or moderate malnutrition
2. Newborn infants
Water-soluble vitamins
Readily excreted
Often are coenzymes in the pathways for energy generation
Dietary sources: milk, meat, egg and seed products
1. Thiamine (B1)
2. Riboflavin (B2)
3. Niacin (B3)
4. Biotin (B7)
5. Pantothenic acid (B5)
Thiamine (B1)
Function:
1. Energy metabolism
- Coenzyme for oxidative decarboxylase reactions (e.g. Pyruvate DH, α-ketoglutarate DH)
2. Other metabolism
- Trans-ketolase in pentose phosphate pathways
Deficiency:
Mild Deficiency: Loss of appetite, nausea, mental depression, peripheral neuropathy, irritability,
fatigue
Moderately severe deficiency:
1. Wernicke-Korsakoff Syndrome
- Mental confusion, ataxia, ophthalmoplegia (loss of eye coordination) selective neuronal damage
and severe memory loss
- Commonly seen in chronic alcoholics
Riboflavin (B2)
Deficiency
- Angular cheilitis
- Stomatitis
- Glossitis
Niacin (B3)
Moderate deficiency
Pellagra
Function:
(i) Used to synthesize CoA to transport carbon atom within the cell
(ii) CoA important for biosynthesize of fatty acids and cholesterol
Deficiency symptoms:
Symptoms: Severe intellectual disability, self-injurious behaviours such as biting their lips and fingers,
involuntary movements such ass spasticity and dystonia, and the presence of uric acid crystals in
urine leading to gout-like symptoms.
Diagnosis: Based on clinical presentation and confirmed by genetic testing to identify mutations in
the HPRT1 gene.
Phenylketonuria (PKU)
Biochemical Deficiency: Mutation in the gene for hepatic enzyme phenylalanine hydroxylase (PAH),
rendering it non-functional, which responsible for converting the amino acid phenylalanine into
tyrosine. When PAH activity is reduced, phenylalanine accumulates and is converted into
phenylpyruvate.
Symptoms: Without treatment, individuals with PKU may develop intellectual disability, seizures,
behavioural problems, and a musty odour due to accumulation of phenylalanine and its breakdown
products in the body.
Diagnosis: Screening of PKU can be performed by using Guthrie test, immunoassays using
fluorometric or photometric detection, or amino acid measurement using mass spectrometry.
Confirmatory diagnosis involves measuring phenylalanine levels in blood plasma and genetic testing
to identify mutations in the PAH gene.
Symptoms: Drowsiness, lack of energy and diarrhoea, and also lead to risk of complications such as
seizures, breathing difficulties and even coma and sudden death.
The Guthrie test is a semiquantitative assay designed to detect elevated blood levels of the amino
acid Phe, using the ability of phenylalanine to facilitate bacterial growth in a culture medium with an
inhibitor. A drop of blood is usually obtained by prickling the heel of a newborn infant on the 6 th or 7th
day of life. The blood is collected on a piece of filter paper and sent to a central laboratory. A small
disk of filter paper is punched out and placed on an agar plate containing Bacillus subtilis and B-
2thienylalanine. Each gel holds 60-80 disks. The agar gel is able to support bacterial growth but B-2-
thienylalanine inhibits bacterial growth. However, in the presence of extra phenylalanine leached
from the impregnated filter paper disk, the inhibition is overcome and the bacteria grow. Within a
day, the bacteriala growth surrounding the paper disk is visible to the eye. The amount of growth,
measured a the diameter of the colony, is roughly proportional to the amount of phenylalanine in
the serum. The result is read by comparing the diameter of each sample disk’s colony with standard
phenylalanine content included on each large plate.