RESPIRATORY SYSTEM MODULE

Biochemistry of pulmonary Surfactant

By Meaza . A (BSc, msc)
2014
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General Objectives
At the end of teaching, students are expected to:
Define the pulmonary surfactant
Explain surfactant composition

Describe the functions of surfactant

Describe metabolisms of surfactant

List diseases of surfactant deficiency

Contents of the chapter

1. Definition of pulmonary surfactant
2. Composition of surfactant

3. Functions of surfactant
4. Metabolisms of surfactant
5. Diseases of surfactant deficiency

History
Research on surfactant goes back to 1929, von Neergaard

published the first paper about the surface tension existing

between the air-water interface of lungs is important factor for
the recoil of lung and reduced surface tension facilitates
respiration.
He found that the surface active agents reduce the resistance

produced by the presence of surface tension.

In 1946 Thannhauser and co-workers reported that lung tissue

has a remarkably high content of the lipid dipalmitoyl lecithin

(current name, dipalmitoyl-phosphatidylcholine)
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Anatomic Aspects of the Lung

The lung is a large organ (~6% of the body volume) with a

large inner surface, continuously in contact with the

environment.
Mammalian lungs are membranous sacs, divided into

alveoli, small sacs that vastly increase the surface area

available for gas exchange.
The lungs contain about 300 million alveoli and serve as

primary sites for exchanging of O2 and CO2.

THE ALVEOLI
Have walls made up of: pneumocytes I and II epithelial cells
and alveolar macrophages
Pneumocytes I:- cover 95% of the alveolar surface area and are
major lining cells where gas exchange takes place
Pneumocyte II cells:- it consists of lamellar bodies of 0.2-2 m
diameter, which comprise 18-24% of the cytoplasm.
These lamellar bodies are the storage form of lung
surfactants

Alveolar macrophages:- The third type of cells present in

the alveoli , involved in removal of surfactant
particles, and microorganisms invading from the air.
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The alveolar pneumocytes- The epithelial cells covering the

alveoli are called alveolar pneumocytes and are two types:
alveolar type I and alveolar type II .
They can be distinguished morphologically and functionally.
Alveolar type I cell are flat squamous cells originating from the
basement membrane and covering 95% of the alveolar
surface area. They possess fewer cell organelles and are
metabolically less active.
Alveolar type II cells are cuboidal cells covering only 5% of
the alveolar surface area. They possess large number of
organelles and are metabolically active.
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Alveolar type II cells are small cells positioned at the

corners or thickenings of the alveoli so that their
morphology does not hinder to gaseous exchange.
These cells are rich in membrane bound organelles
including lamellar bodies which are the sites of secretion
and storage of pulmonary surfactant.
In addition, they have role in alveolar fluid balance,
immuno-regulation and host defense.

Structure of Alveolus

Concept of Surface Tension-Surfactant

Gas exchange in the lung takes place in the alveoli.
Oxygen diffuses from the alveoli to the capillaries, and
carbon dioxide leaves the capillaries and diffuses into the
alveoli.

Surface tension is an effect within the surface layer of a liquid

that causes that layer to behave as an elastic sheet
OR
Surface tension can be defined as cohesive force of attraction
experienced by the molecules present at the interface of
two media.

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The surface tension of the moist inner surface originates

from the attraction between the molecules in a fluid and is
responsible for the tendency to make the contract.

This tendency is minimized by the presence of a surface

active agent or surfactants. These substance that reduces
the surface tension on the inner surface of the alveoli to
very low values.

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What is Surface Tension?

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Pulmonary Surfactant
Definition
Lungs offer a large surface area that comes directly in
contact with air for gaseous exchange in to body fluid.
The surface tension at the gaseous-liquid interface of
lung is reduced by a substance, which is found in the
lungs of all mammals, is called lung surfactant.
It is a heterogeneous mixture of lipids and proteins that
forms a stable monolayer at gaseous-aqueous interface
Serves as good mediator to settle dispute between
two phases which are not friends
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Surfactant
Pulmonary surfactant is a surface-active lipoprotein
complex (phospholipoprotein) formed by type II alveolar
cells.
The proteins and lipids that make up the surfactant have
both hydrophilic and hydrophobic regions.
By adsorbing to the air-water interface of alveoli
hydrophilic head groups in the water and the hydrophobic
tails facing towards the air, the main lipid component of
surfactant, dipalmitoylphosphatidylcholine (DPPC),
reduces surface tension.

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Surfactant
Surface tension is inversely proportionate to surfactant
concentration per unit area.
Inspiration/Inhalation--> Expansion of alveoli--> increase
surface area of fluid --> dispersion of surfactant--> decrease
surfactant effect--> increase surface tension for the prevention
of alveolar over distension and rupture.
Expiration /Exhalation -- Recoil of alveoli to smaller size--->
decrease fluid surface area--> condensed surfactant-->
increasing surfactant activity--> decrease surface tension
these is important for prevention of lung collapse.
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Surfactant
Surfactant is produced by the alveolar type II cells in the
lung.
Two major surfactant pools can be distinguished:
The extracellular surfactant compartment : surfactant
that is secreted into the alveolar space.
An intracellular surfactant compartment: consists of the
lamellar bodies in the alveolar type II cells. Their
function is storage of surfactant before it is released
into the alveolar space

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The lamellar bodies

Lamellar granules (lamellar
bodies): are lysosome like
membrane bound
organelle found in type
II pneumocytes.
Lamellar bodies are important
for storage and secretion sites for
surfactant.
Lamellar bodies fuse with the
cell membrane and release their
contents into the extracellular
space
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how does surfactant work ?

Water :
Oil :

Hydrophobic
Lipophilic

Hydrophilic
Lipophobic

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The Influence of Surfactants on

Surface Tension

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Composition of Surfactants
Surfactant is comprised of :
- 85% Glycerophospholipid
-5% Cholesterol (neutral lipids), and
-10% Protein.
The principal glycerophospholipid in surfactant is DPPC
(Dipalmitoylphoshatidylcholine).
Although most of surfactant consists of lipids, it comprises
approximately 10% protein.
These proteins can be divided into two groups:
a. The hydrophilic surfactant proteins SP-A and SP-D,
b. The hydrophobic surfactant proteins SP-B and SP-C.
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- Surfactants are:
Produced, secreted, and recycled by Type II pneumocytes
- The major phospholipid components are:
1. Dipalmitoylphosphatidylcholine, DPPC ( 30 45 % )
2. Unsaturated phosphatidylcholine (25 35%)
- Phosphatidylcholine (PC) is the main phospholipid class,
with dipalmitoylphosphatidylcholine (DPPC) the
commonest subtype.
- Monolayers of DPPC are crucial in the maintenance of
surface tension at near zero levels.

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Surfactant lipid biosynthesis

The biosynthetic pathway for the pulmonary surfactant
lipids and the membrane lipids is the same.
The surfactants is mainly synthesized in the alveolar type
II cells and few other cells of the respiratory air ways.
The biosynthesis of lipids constituting the surfactant is
basically the biosynthetic pathway for PC, PG and PI
biosynthesis from the precursors like
dihydroxyacetonephosphate(DHAP), glyceraldehyde-3phosphate, phosphatidic acid, choline, some acyl
derivative's of these.

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Hydrophilic
head

Hydrophobic
tails

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DPPC (Dipalmitoyl phoshatidyl choline)

This is a phospholipid with two 16-carbon saturated
chains and a phosphate group with quaternary amine
group attached.
The DPPC is the strongest surfactant molecule in the
pulmonary surfactant mixture.
Phosphatidylcholine is an exception, biological
phospholipids are synthesized with a saturated fat at the
R1 position and an unsaturated fat at the R2 position.

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Dipalmitoylphosphatidylcholine :
(consisting of two palmitic acids)

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Synthesis of DPPC
* Two main pathways for lung PtdCho synthesis
* Both pathways have the potential to produce DPPtdCho.
(A)de novo PtdCho synthesis involves three major enzymes:
(i) Choline kinase (CK),
(ii) CTP: phosphocholine cytidylyltransferase (CCT), and
(iii) Cholinephosphotransferase (CPT1).
(B) The remodeling pathway involves the reacylation of a PtdCho
degradation product (LysoPtdCho) by acyl CoA:
lysophosphatidylcholine acyltransferase (LPCAT1) for the
synthesis of DPPtdCho.

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Synthesis of DPPC from DHap

Glucose
NAD+ NADH

glycerol-3-phosphate

Glycogen

DHAP

palmitoyl-CoA
CoASH

Choline

palmitoyl-G3P
palmitoyl-CoA

ATP

CoASH

ADP

dipalmitoylphosphatidic acid

phosphocholine

H2O

CTP

Pi

PPi

dipalmitoylglycerol

CDP-choline
CMP

DPPC

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The enzyme involved in the synthesis of surfactant from DHAP.

The surfactant phospholipids are Synthesized from
dihydroxy acetonephosphate (DHAP) using as precursor
for the surfactant DPPC.
DHAP is converted in to glycerol-3- phosphate by enzyme
DHAP dehydrogenase, in the presence of NAD+ as
coenzyme. Glycerol-3- phosphate converted in to
palmitoyl glycerol-3-phosphate, these step catalyzed by
glycerol-3- phosphate acyl transferase by transferring acyl
group.
Then palmitoyl glycerol-3-phosphate converted in to
dipalmitoyl phosphatidic acid by palmitoyl glycerol-3phosphate acyl transferase.
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Now phosphatidic acid is synthesized then to degrade

phosphatidic acid by using of phosphatidic acid
phosphatase and produced DAG (diacylglycerol) that
means dipalmitoylglycerol.
Finally these, diacylglycerol bind with active choline
(CDP-choline) and produced
dipalmitoylphosphatidylcholine (DPPC), these reaction
catalyzed by choline phosphotransferase.
These phospholipids are responsible for the reduction of
surface tension of water by associated with surfactant
proteins.
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Regulation of lipid biosynthesis

1. Enzymatic regulation: the biosynthesis of PC is mediated by

sequential actions of different enzymes.

The reactions catalyzed by phosphpocholine
citidyltransferase (CCT) is the rate limiting step for PC
synthesis. Therefore, modulating CCT serve as regulatory
mechanism for the biosynthesis of PC.
CCT is an enzyme present as active and inactive forms.
Translocation of CCT from inactive form to active is
promoted by low level of PC or more amounts of fatty acids
or diacylglycerols (DAG, precursors of PC) in the cells.

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2. Hormonal regulation: various hormones like

catecholamine's, and insulin have been shown to increase
the surfactant lipid biosynthesis.
The biosynthesis can be increased by either by increasing
enzyme activity or by modulating cellular metabolism to
increase the availability of precursors like DAG and other
lipids for enzyme (increasing the substrate availability and
enzyme activity indirectly)
Therefore, hormonal regulation of surfactant biosynthesis
assumes paramount importance for the development and
maturation of lungs.
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SP (Surfactant Proteins)
1. Hydrophilic Surfactant Proteins
Two hydrophilic surfactant proteins have been isolated, SP-A
and SP-D.
These two are large glycosylated proteins and belong to a
subgroup of mammalian lectins called collectins.
The genes encoding SP-A, and SP-D are transcribed in the
nucleus of alveolar type II epithelial cells, translated into
nascent polypeptides in the endoplasmic reticulum (ER).
The translated product of these gene undergoes glycosylation
in the Golgi bodies and multivesicular bodies to form the
functionally mature proteins.

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The other hydrophilic collagenous glycoprotein found in

bronchoalveolar lavage is SP-D
It is has different functions and the production of SP-D is not
exclusively in the lung; SP-D mRNA is also found in gastric
tissue

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2. Hydrophobic Surfactant Proteins

Two hydrophobic surfactant proteins are known:
SP-B and SP-C.
These proteins are expressed in alveolar type II cells and
bronchiolar epithelial cells.
The translated product of these gene undergoes glycosylation
in the Golgi bodies and multivesicular bodies to form the
functionally mature proteins.
The mature proteins are transported to the lamellar bodies
along with the various phospholipids by transporter (ABCA3).
It is important for the aggregation of lipids in to the lamellar
body and facilitation of the formation of surfactant monolayer
at the interface.
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SP-C
The second member of the group of hydrophobic surfactant

proteins is SP-C

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Surfactant secretion
Various physical and chemical stimuli can induce
surfactant secretion. The physical stimuli include
mechanical stretch or contraction of the alveolar type II
cells during breathing.
The chemical agents include catecholamine, and cAMP.
Up on stimulation the lamellar bodies are translocated to
the apical side of alveolar type II cells where they
undergo exocytosis to release their contents in to the
extracellular matrix.

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The secreted form of surfactant as such is non functional

but after secretion it undergoes structural/conformational
changes to give rise to an intermediary lattice structure
known as tubular myelin.
The tubular myelin then forms surfactant monolayer by
adsorbing the lipid components in to the air-water
interface. This monolayer is the functionally active form
of surfactant and has surface-active properties.

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Surfactant recycling
After secretion and incorporation in to monolayer,
surfactant undergo dissociation in to its component.
Most of the components are recycled back through
receptor mediated endocytosis in to alveolar II cells while
some components are cleared by the airway lining and
alveolar macrophages.
The recycling helps to maintain surfactant pools at the
alveolar level and conserve energy required for
synthesizing the components again.

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For TODAY. . .

Functions of Surfactant
These includes:
To increase pulmonary compliance.
To prevent atelectasis (collapse of the lung) at the end of
expiration.
To keep alveoli dry
To regulate the size of alveoli
To play roles in pulmonary host defense

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Compliance
Compliance is the ability of lungs and thorax to expand.
Lung compliance is defined as the volume change per unit
of pressure change across the lung.

Measurements of lung volume obtained during the

controlled inflation/deflation of a normal lung.
However, surfactant decreases the alveolar surface
tension, as seen in cases of premature infants suffering
from infant respiratory distress syndrome.
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Pulmonary surfactant thus greatly reduces surface tension,

increasing compliance allowing the lung to inflate much
more easily, thereby reducing the work of breathing.
The lung's compliance decreases and ventilation decreases
when lung tissue becomes diseased.

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Alveolar size regulation

As the alveoli increase in size, the surfactant becomes
more spread out over the surface of the liquid.
This increases surface tension effectively slowing the rate
of expansion of the alveoli.
This also helps all alveoli in the lungs expand at the same
rate, as one that expands more quickly will experience a
large rise in surface tension slowing its rate of expansion.
Surfactants reduce surface tension more readily when the
alveoli is smaller because surfactants are more
concentrated.
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Prevent fluid accumulation and keep airways dry

Surface tension forces also draw fluid from capillaries to
the alveolar spaces.
Surfactant reduces fluid accumulation and keeps the
airways dry by reducing these forces.

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Host defense
Surfactant immune function is primarily attributed to two
proteins: SP-A and SP-D.
These proteins can bind to sugars on the surface of
pathogens and thereby opsonize them for uptake by
phagocytes.
Surfactant degradation or inactivation may contribute to
enhanced susceptibility to lung infection.

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Pulmonary surfactant metabolism and

homeostasis
The genes encoding SP-A, Pro-SP-B and Pro-SP-C are
transcribed in the nucleus of alveolar type II epithelial cells,
translated into nascent polypeptides in the endoplasmic
reticulum (ER) and processed in the Golgi.
SP-B and SP-C are assembled into lamellar bodies along
with surfactant phospholipids, the transport of which may
be regulated by the ABCA3 transporter molecule, which is
found in the limiting membrane of these organelles.

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SP-A is secreted via non-lamellar body secretory vesicles

(Multivesicular body).
Following exocytosis of lamellar bodies and secretory
vesicles into the alveolar surface liquid (Secretion), lamellar
bodies assemble into structures known as tubular myelin.
Phospholipids from these extracellular structures move to
form a continuous surfactant film that lines the alveolar
spaces and airways with polar heads oriented toward the
liquid and acyl chains toward the air.

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Surfactant Inactivation
The interaction between the lipids and proteins is very

critical for the functioning of surfactant. Different factors

affect the active lipid/protein complexes
Inactivation caused by:
1. The addition of small particulate entities from the
interface,
2. Changes in lipid/protein organization
3. Genetic alterations

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4. The exposure of surfactant to potent environmental

oxidants such as ozone
5. Phospholipases or proteolytic enzymes can cleave
surfactant components

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Synthesis and secretion of the protein and phospholipid

components of surfactant proceed via separate
pathways
SP-B and SP-C traffic through the Golgi and
multivesicular body (MVB) to the lamellar body
In contrast, surfactant phospholipids may traffic directly
from the ER to the lamellar body

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Extracellular Surfactant Metabolism

After secretion, surfactant is transformed into specific

structures, called tubular myelin, from which insertion of

phospholipids into an air-liquid interface is thought to
take place
The phospholipid molecules are found with their

hydrophobic fatty acid chains up in the air and their

(polar) headgroups in the liquid.

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Extracellular Surfactant Metabolism

Phospholipid adsorption is required to ensure molecular
occupation of the air-water interface during inflation of the
lung.
Not only is the formation of the monolayer stimulated by
the hydrophobic proteins, but it has been reported that SP-B
may also reduce the surface tension by increasing the
stability of the phospholipid layer.

The composition of the monolayer is also an important

factor in the adsorption of the surface-active material into
the monolayer
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During expiration: the surface tension at the air-water

interface of the lung is reduced.
To reach a low surface tension, the monolayer becomes
enriched in DPPC.

During the next inhalation, and expansion of the surface area

of the alveoli, the surfactant components are scattered and
causing high surface tension.
During this process surfactant components are lost from
the interface and taken back into the type II cell for
recycling.

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Regulation of lung surfactant production and

clearance
Agents that stimulate adenylate cyclase and increase
cytosolic cyclic AMP levels such as catecholamine's and
glucagon and also gas entering in to the lungs and alveolar
stretch (inspiration) stimulate the secretion of surfactant by
type II cells.
A hormones including insulin have been reported to
enhance the synthesis of glycerophospholipids of lung
surfactant.

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There are several possible pathways by which the

secreted surfactant may eventually be cleared from the
alveoli including
- Ingestion by alveolar macrophages,
- Movement up to the airways,
- Enzymatic degradation at the alveolar surface,
- Uptake by type II cells and other cells.

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Clinical importance
The decrease in surfactant level/ function is associated
with a number of disease like infant respiratory distress
syndrome /IRDS/, adult respiratory distress syndrome /
ARDS/, lung proteinosis, obstructive lung disease.
There are two mechanisms involved in the impairment of
surfactant biosynthesis and functioning, these are:
1. Genetic factors include mutations in genes coded for
surfactant proteins and proteins that are involved in
transport/transporter proteins.

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Mutations in genes coding for SP-A, SP-B,SP- C and SP-D

and surfactant transporting proteins are associated with
respiratory distress syndrome
Mutations in genes coding for SP-A and SP-D proteins are
associated with immunological deficiencies and may lead
to the development of respiratory infections.
For example: Mutations of genes coding for SP-B, SP-C
and ABCA3 transporter.

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Disorders of SP-B gene (SFTPB)

Mutations in the human SP-B gene (SFTPB ) cause
surfactant dysfunction and lethal respiratory distress in fullterm infants.
Hereditary SP-B deficiency is an autosomal recessive
disease caused by mutations in the SFTPB gene that is
located on human chromosome 2.
Mutations in the SFTPB gene result in either lack of SP-B
mRNA or production of abnormal SP-B proproteins that
result in misprocessed protein that disturbs synthesis of the
active SP-B protein.

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In addition, SP-B is required for the normal routing and

packaging of surfactant lipids and surfactant protein C (SPC) in type II epithelial cells of the lung
Thus, deletion of SP-B also results in the absence of SP-C in
the airspaces.
Generally, the mutations in genes coding SP-B cause lethal
respiratory distress following birth.

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Disorders of the SP-C gene (SFTPC)

SP-C is a small hydrophobic protein that plays an
important role in surfactant function of lung homeostasis.
A single gene encoding SP-C (SFTPC ) is located on
human chromosome 8.
Hereditary SP-C deficiency, mutations in the SFTPC gene
cause both acute and chronic pulmonary disease in
humans.
Both lack of SP-C and mutations in the gene encoding SPC (SFTPC ) have been associated with acute and chronic
lung disease in infants and adults.

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Disorders of the ABCA3 transporter gene

Mutations in the ABCA3 transporter is one cause of acute
RDS newborn infants, and the cause of chronic lung
disease in older individuals
ABCA3 is a 1704 amino acid, multiple transmembrane
protein of the family of ATP-binding cassette (ABC)
transporters, and involved in the multiple drug resistance.

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ABCA3 is expressed in type II epithelial cells of the lung,

being detected at the limiting membranes of lamellar
bodies in type II epithelial cells.
Lung disease associated with mutations in the ABCA3
gene are inherited in an autosomal recessive manner.
The mutation of genes of these transporter proteins are
associated with respiratory distress syndrome.

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2. Metabolic factors includes decreased secretion of

surfactant, Proteolytic/ lipolytic cleavage of surfactant
components or oxidative damage to surfactant components.

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Pathophysiology of diseases associated

with surfactant insufficiency
Respiratory Distress Syndrome
*Respiratory distress syndrome is associated with surfactant
deficiencies. It is characterized by increased work of
breathing, impaired gas exchange, decreased compliance,
atelectasis, and other complications.
It is very common in infants, where it is called infant
respiratory distress syndrome (IRDS).
IRDS is characterized by the immature lungs and failure to
produce sufficient amount of surfactant.

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Genetic or acquired factors are implicated for the

development of IRDS.
These factors include the mutations in genes coding for
the surfactant proteins and transporter proteins. Non
genetic factors are gestational age, maternal health, and
lung maturity.
IRDS they fail to form tubular myelin because of
deficiency of surfactant proteins and phospholipids that
are essential for the adsorption in to functional
monolayer.

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when there is insufficient surfactant because of these

factors, causes the alveoli collapse during exhalation.
Later causing decreased oxygenation of the collapsed alveoli
and respiratory distress in the neonate.
This condition was formerly known as hyaline membrane
disease.
The infant develops rapid, difficult respirations, within a few
hours of delivery.
Amniotic fluid testing can reveal decreased phosphatidyl
glycerol or other phospholipids.

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In adults, RDS is called Adult respiratory distress

syndrome (ARDS). It is a complex disorder which is
characterized by deficiency of surfactant.
The major pathophysiological mechanisms leading to
surfactant deficiency is the accumulation of fluid in the
alveolar space and/ or decreased pulmonary fluid
clearance resulting in pulmonary edema.

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Pulmonary edema is rich in plasma proteins and

proteolytic enzyme that degrade surfactant proteins.

In addition, the tissue damage are altered the functional

and compositional properties of surfactant lipids and
proteins.

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Pulmonary alveolar proteinosis

It is a rare lung disease, caused by the abnormal
accumulation of surfactant contents occurs within
the alveoli
There is increased amount of surfactants but there is
impaired formation of tubular myelin.

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Obstructive lung disease

Various obstructive lung disease like chronic obstructive
pulmonary disease (COPD), and asthma are associated with
decreased surfactant function.
Obstructive lung disease are characterized by chronically
poor airflow, release of various proteins, proteolytic
enzymes, inflammatory mediators, and reactive oxygen
species.
All these chemical agents decrease surfactant availability at
the interface by decreasing its synthesis or functional
capacity
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Therapeutic agents used in

surfactant deficiency states
The therapeutic strategies used for decreased surfactant
or surfactant function can be divided in to endogenous
and exogenous agents.
The endogenous agents include hormones and other
pharmacological substances that increase surfactant
synthesis or secretion in the lungs whereas
Exogenous agents include direct administration of
surfactant or surfactant proteins.

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Summary and conclusion

Pulmonary surfactant, a mixture of lipids and proteins, is
very important for proper lung function. It decreases
surface tension at the alveolar interface, prevents collapse
of alveoli during expiration.
The maintenance of lung compliance, host defense and
other immunoprotective are the functions of pulmonary
surfactant.
The surfactant is synthesized and secreted by alveolar
epithelium called type II alveolar pneumocytes. These cells
contain membrane bound organelles called lamellar body.

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The components of the surfactant are synthesized

separately and are packed in to the lamellar bodies.
Any genetic or metabolic abnormalities in surfactant
homeostasis can result in surfactant deficiency.
Deficiency of surfactant is seen in disease like respiratory
distress syndrome, obstructive lung disease, e.t.c.
A combination of surfactant (natural or synthetic) and
hormones or other pharmacological substances associated
with surfactant homeostasis are useful in treating these
diseases.

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