Biochemistry of

Respiratory System
1,2Marhaen Hardjo
1Head of Biochemistry Department, Medical Faculty of Hasanuddin University
2Director of Stem Cell Center Hasanuddin University Hospital

DEPARTMENT OF BIOCHEMISTRYY
MEDICAL FACULTI OF
HASANUDDIN UNIVERSITY
Tujuan Instruksional Umum:

Setelah menyelesaikan mata kuliah ini,

mahasiswa akan mampu mengenali dan
menganalisis aspek Biokimia berbagai proses
tubuh yang terkait dengan mekanisme dasar
sistem pernapasan untuk mempertahankan
fisiologis tubuh yang optimal, dan mampu
mengintegrasikan dan menerapkannya kasus
kasus penyakit terkait sistem pernafasan
Tujuan Instruksional Khusus
Setelah mengikuti kuliah ini, mahasiswa mampu:
I. Memahami Biokimia sistem pernapasan terkait:
1.Respirasi Sel (internal respiration)

2.Peran oksigen dalam proses metabolisme dan

oksigenasi jaringan
3.Faktor yang berperan dalam oksigenasi jaringan
(Saturasi oksigen)
4.Fungsi Regulasi dan Kompensasi Paru2 dalam
Keseimbangan Asam Basa
5.Hb: Struktur, fungsi dari derivat dan variannya

6.Dasar Biokimia Analisa Gas Darah

II. Mengintegrasikan dan menerapkan aspek Biokimia

proses pernapasan dengan kasus-kasus penyakit terkait
sistem pernafasan
Pokok Bahasan 1

Respirasi Sel
RESPIRASI SEL
 Merupakan jalur pelepasan energi dengan pembentukan ATP
 Terbagi atas 2 keadaan :
☺Aerobik → perlu O2 ; pada organisme multiseluler kompleks
dan aktif ; C6H12O6 + 6 O2  6 CO2 + 6 H2O ; ATP yang
dihasilkan lebih banyak dari respirasi anaerob (sekitar 36
ATP lebih atau hingga 38 ATP untuk setiap molekul glukosa)
☺Anaerobik → tidak perlu O2 ; organisme uniseluler dan
tingkat rendah ; ATP yang dihasilkan jumlah lebih sedikit,
yaitu 2 ATP untuk setiap molekul glukosa
 Respirasi sel aerob terdiri atas 3 tahap
1. Glikolisis
2. Tahap persiapan dan siklus Krebs
3. Fosforilasi transport elektron
 Respirasi sel anaerob terdiri atas 2 keadaan :
Jalur fermentasi
Transport elektron anerobik
Gambaran umum respirasi aerobik
GLIKOLISIS
 Berlangsung di sitoplasma ; glukosa → glukosa 6-fosfat
→ 2 piruvat
 Terdiri atas 2 tahap :
1. Membutuhkan energi (energy requiring step) → transfer
gugus fosfat dari 2 molekul ATP pada 1 molekul glukosa →
fosforilasi
2. Melepaskan energi (energy releasing step) → fosforilasi
pada tingkat substrat
fruktosa 1,6-bifosfat (molekul glukosa yang teraktifasi) →
PGAL (fosfogliseraldehid) → molekul intermediat yang tidak
stabil (ada gugus fosfat) → gugus fosfat dilepas pada ADP
membentuk ATP → dihasilkan 4 molekul ATP & NADH

a. Energi netto : 2 mol ATP

b. NAD+ + e- + H+ → NADH
↓
koenzim nicotinamide adenin dinucleotide → reuse
Glikolisis
TAHAP PERSIAPAN DAN SIKLUS KREBS
 Berlangsung di mitokhondria
 Glukosa dipecah secara sempurna → CO2 + H2O
 Ciri-ciri yang mencolok pada tahap ini : transfer e- & H+ dengan
melibatkan banyak koenzim (NAD+ & FAD+) untuk membentuk
NADH dan FADH2 dalam jumlah yang banyak.
Sehingga terdapat 3 fungsi : (1) terangkutnya elektron dan
hidrogen melalui NAD+ dan FAD+, sehingga terbentuk NADH
dan FADH2 ; (2) terbentuknya 2 ATP pada fosforilasi tingkat
substrat dan (3) pengaturan kembali bentukan intermediat
siklus Krebs menjadi oksaloasetat
 Tahap persiapan (dekarboksilasi oksidatif)
Enzim (koenzim A) memindahkan 2 atom C dari piruvat →
asetil ko-A
 Siklus Krebs (siklus asam sitrat/TCA cycle)
Oksaloasetat (4 atom C) sebagai titik awal masuk siklus +
piruvat (2 atom C) → asam sitrat (6 atom C) → dst jalur
siklik enzim-enzim & koenzim
 Jumlah energi yang dihasilkan dengan bantuan koenzim :
glikolisis 2 NADH
dekarboksilasi oksidatif 2 NADH
siklus Krebs 6 NADH + 2 FADH2
---------------------------- +
10 NADH + 2 FADH2
 Jika NADH dikonversi menjadi 3 ATP dan FADH2 menjadi 2
ATP maka jumlahnya adalah 34 ATP
 Jumlah total net ATP yang dihasilkan pada respirasi ini setelah
konversi bentukan energi di atas pada tahap ke-3 akan
bervariasi (antara 36 ATP hingga 38 ATP) tergantung pada
perubahan konsentrasi reaktan, bentukan intermediat atau
produknya.
 Tahap kedua dari respirasi aerob :
siklus Krebs dan reaksi yang mendahuluinya.
FOSFORILASI TRANSPORT ELEKTRON
 Terjadi dalam matriks (inner compartment/ruang dalam) &
ruang intermembran (outer compartment/ruangan luar) ;
terdapat sistem transport elektron dan sintase ATP →
berinteraksi dengan e- dan H+ dari koenzim-koenzim →
fosforilasi transport elektron
 e- masuk dalam sistem transport elektron pada membran
dalam sehingga menyebabkan adanya gradien konsentrasi &
elektrik padanya H+ (ruangan dalam) → ↑↑↑ (ruangan
luar) H+ (ruangan luar) → ↓↓↓ (ruangan dalam) dengan
membentuk ATP dan H2O (O2 sebagai akseptor terakhir dari H+
dan e-
 TEM dan sketsa dari mitokhondria.
(b) Daerah-daerah fungsional dalam mitokhondria
JALUR FERMENTASI
 Terjadi dalam sitoplasma & pada organisme sel-sel prokaryotik
& Protista
 Dimulai glikolisis : glukosa ------------------> 2 mol piruvat
2 ATP & 2 mol NADH
tahap akhir regenerasi NAD+ (koenzim pada glikolisis)
 Terbagi atas 2 jenis :
Fermentasi laktat : pada Bakteri dan sel-sel otot
piruvat + e- + H+ → laktat + NAD+↑

dari NADH
Fermentasi alkohol :
piruvat --------> asetaldehid ------> alkohol (seperti etanol)
enzim
e- + H+ dari NADH
Fermentasi laktat
Fermentasi alkohol
TRANSPORT ELEKTRON ANEROBIK
 Oleh organisme prokaryotik yang berperan dalam siklus global
dari sulfur, nitrogen & elemen lain untuk suplai nutrien ke dalam
ekosistem
Seperti : Archaebacteria & Eubacteria tertentu
 Terjadi aliran e- pada sistem transport di membran plasma
 Jumlah energi yang dihasilkan bervariasi tetapi dengan jumlah
yang sedikit.
SUMBER ENERGI ALTERNATIF
 Terdapat beberapa sumber energi alternatif pada manusia
selain glukosa yang harus difosforilasi terlebih dahulu setelah
masuk dalam sel sehingga tidak dapat keluar sel lagi
 Glikogen (polisakarida) yang dipecah menjadi glukosa jika
dibutuhkan dan akan dibentuk kembali jika kadar glukosa
dalam darah meningkat sementara konsentrasi ATP dalam
sitosol sel masih besar.
 Lemak akan dipecah lebih dulu menjdi lemak dan gliserol
sebelum masuk jalur pelepasan energi
 Protein akan dipecah menjadi asam-asam amino sebelum
masuk jalur tersebut
Sumber energi alternatif dalam tubuh manusia
 Pokok Bahasan 2

Peran oksigen dalam proses

metabolisme dan oksigenasi jaringan
 Aerobic versus Anaerobic Metabolism
 Heterotrophs
 Aerobes: Use molecular oxygen as the final
electron acceptor
 Anaerobes: Use other molecules as final electron
acceptor
 Energy yield much lower ATP yield
 When oxygen acts as the final electron acceptor
(aerobes):
 Almost 20 times more energy is released than if
another acceptor is used (anaerobes).
 Advantage of aerobic metabolism:
 Smaller quantity of food required to maintain
given rate of metabolism.
 Aerobic Respiration
 In aerobic respiration, ATP forms as electrons
are harvested, transferred along the electron
transport chain and eventually donated to O2
gas.
 Oxygen is required!
 Glucose is completely oxidized.
 C6H12O6 + 6O2 6CO2 + 6H2O + energy (heat
Glucose Oxygen Carbon Water or ATP)
Dioxide
Cellular Respiration - 3 Stages
 Food is digested to break it into
smaller pieces – no energy
production here.
 Glycolysis – coupled reactions
used to make ATP.
 Occurs in cytoplasm

 Doesn’t require O2

 Oxidation – harvests electrons

and uses their energy to power
ATP production.
 Only in mitochondria

 More powerful
Anaerobic Respiration
 Anaerobic respiration occurs in the absence of
oxygen.
 Different electron acceptors are used instead of
oxygen (sulfur, or nitrate).
 Sugars are not completely oxidized, so it doesn’t
generate as much ATP.
 Pokok Bahasan 3
Faktor yang berperan dalam oksigenasi
jaringan (Saturasi oksigen)
The critical function of the pulmonary system
is to facilitate respiration

Respiration comprises

Lung uptake and delivery of O2 to tissues

Lung removal of CO2 from tissues

 Why Tissue Oxygenation?

O2 is required for the production of energy (as ATP) during

oxidative metabolism in the mitochondria

CO2 is a toxic by-product of the of the metabolism of

CHO and fats
What factors facilitate Tissue oxygenation?

Alveolar ventilation and function

Pulmonary and systemic blood flows

O2 binding in alveoli and release in tissue (Hb)

How can we assess tissue oxygenation / tissue hypoxia?

Clinically
Central cyanosis >5g/L deoxygenated Hb (SaO2 < 67%)
Hypotension
Organ dysfunction e.g. ARDS, ARF
Mental obtundation

Plasma/Blood lactate

Arterial O2 Saturation (SaO2)

-limitations
-pulse oximeter

PaO2 (arterial partial pressure of O2)

- Still an essential index of tissue O2 supply
 Limitations of SaO2

No information about respiratory ventilation – require ABG

Large fall in PO2 might cause only a small fall in SaO2

e.g. SaO2 90% could still reflect a large fall in PO2

O2 dissociation curve
PaCo2 is a useful means of assessing ventilation

Clinically:

•Rate and depth of respiration – limited accuracy

PaCO2 (arterial partial pressure of CO2):

•Key assessment of alveolar ventilation

•Increase ventilation lowers PCO2
•Decreased ventilation increases PCO2
 Pokok Bahasan 4
Fungsi Regulasi dan Kompensasi Paru2
dalam Keseimbangan Asam Basa
Disorders of acid-base balance
Three mechanisms are involved in regulating
changes in ECF acid-base balance

Buffers

Respiratory response

Renal response
Buffers act to limit change in acid-base status

Intracellular buffers

- Proteins e.g Hb in red cells

- Bone

Extracellular buffers

- Phosphate (HPO4)

- Bicarbonate buffer (HCO3)

 The Bicarbonate buffer system is uniquely tailored to
regulating acid-base balance

Most extracellular buffers have a limited capacity i.e. become saturated

However in the case of the HCO3 Buffer system

H + HCO3 H2CO3 CO2 + H2O

The end product CO2 can be dissipated via lungs

Thus the HCO3 buffer system is less likely to become saturated

The relationship between the HCO3 buffer and pH can be
predicted by the Henderson-Hasselbach equation

pH = 6.1 + log HCO3/H2CO3

pH = 6.1 + log HCO3/0.03PCO2

pH depends HCO3 (Renal)

upon PCO2 (Lung)

[H+] (nmol/l) = 180 X PCO2/[HCO3] N.B. PCO2 (kPa)

 Regulation of acid-base balance is primarily
dependent on two main organ systems

Respiratory (lungs) – regulates PCO2

•Increased or decreased ventilation

Renal – regulates HCO3

•Reabsorption of HCO3 (proximal tubule)
•Generation of HCO3 (distal tubule – urine pH < 5.5)
•Titratable acidity (HPO4 buffers throughout tubule)

The respiratory response occurs more quickly than

the renal response
The relationship between the HCO3 buffer and pH can be
predicted by the Henderson-Hasselbach equation

pH = 6.1 + log HCO3/H2CO3

pH = 6.1 + log HCO3/0.03PCO2

pH depends HCO3 (Renal)

upon PCO2 (Lung)

[H+] (nmol/l) = 180 X PCO2/[HCO3] N.B. PCO2 (kPa)

Disorders of acid-base balance

Acidosis/Acidaemia ( pH, [H+] )

-Respiratory PCO2

-Metabolic HCO3

Alkalosis/Alkalaemia ( pH, [H+])

-Respiratory PCO2

-Metabolic HCO3
 Compensatory mechanisms exist to limit the extent
of acid-base disturbance and restore pH towards normal

Metabolic acidosis Respiratory alkalosis ( PCO2)

Respiratory acidosis Metabolic alkalosis ( HCO3)

Metabolic alkalosis Respiratory acidosis ( PCO2)

Respiratory alkalosis Metabolic acidosis ( HCO3)

The respiratory response can occur within minutes but the renal
response can take 2-4 days to develop

Full compensation does not occur except in the case of chronic

respiratory alkalosis
 Causes of Respiratory Acidosis

-CNS depression e.g. trauma, drug OD

-Neuromuscular disorders

-Chest wall disease e.g. kyphoscoliosis

-Pleural effusions

-COPD

-Pulmonary oedema
 Causes of Metabolic Acidosis

High anion gap

•Ketoacidosis
•Lactic acidosis
•Toxins e.g. methanol, ethanol,salicylate OD
•Renal failure

Normal anion gap (hyperchloraemic)

•Renal tubular acidosis (Type I, II and IV)
•Early stages of CRF
•Diarrhoea, ureteric diversion (HCO3 loss)
•Ingestions/infusions e.g. HCl
 The Anion Gap may be useful in determining
the cause of acidaemia

AG = (Na + K) – (HCO3 + Cl)

Ref range: 7- 17 mmol/l

Increased AG suggests the presence of circulating anion

e.g ketones, lactate, salicylate
 Causes of Respiratory Alkalosis

-Anxiety/hysteria related hyperventilation

-CNS pathology causing hyperventilation

-CCF/pulmonary oedema

-Salicylates

-Sepsis

-Cirrhosis

-Ventilator induced
 Causes of Metabolic Alkalosis

Chloride/Saline Responsive
GI losses e.g. vomiting, gastric suction, Cl diarrhoea

Chloride/Saline Unrepsonsive
Diuretic therapy
Mineralocorticoid excess e.g. Conn,s syndrome,
exogenous
Cushing’s syndrome
Bartter/Gitelman syndrome

Hypokalaemia is very often associated with the

pathogenesis of metabolic alkalosis
Evaluation of patient with suspected acid-base
disturbance (1)

What is the clinical picture?

-Hx DM, CRF,
-Vomiting, diarrhoea
-COPD,
-Hyperventilating

What are the plasma electrolytes?

-hypo/hyperkalaemia
-Renal failure
-Glucose
-AG
 Evaluation of patient with suspected acid-base
disturbance (2)
Arterial Blood Gases (ABG)
-usually done by trained staff as a point of care test (POCT)

3 basic values provided

• pH or [H+]

• PCO2 (also PO2)

• [HCO3] (derived from H-H equation)

Other values
Base excess – measure of metabolic component
Standard HCO3 – measure of metabolic component
What are the measured components of an ABG?

Component Ref. Range

PO2 (kPa) 11.1-14.1

PCO2 (kPa) 4.4-6.4
pH 7.35-7.45
[H+] (nmol/L) 36-45
HCO3 (mmol/L) 21-31

Standard HCO3 and Base Excess (BE) are measures of “metabolic

component” but give similar information to actual HCO3
A C

pH 7.27 (7.35 – 7.45) pH 7.25

PCO2 2.66 (4.6 – 6.4) PCO2 7.2
HCO3 9 (22-31) HCO3 22

B D
pH 7.05 pH 7.58
PCO2 5.5 PCO2 1.6
HCO3 8 HCO3 19
A = partially compensated metabolic acidosis

B = Uncompensated metabolic acidosis

C = Uncompensated respiratory acidosis

D = partially compensated respiratory alkalosis

 Mixed Acid-Base disturbances

•Implies that there is more than one disorder of acid-base

balance ocurring simultaneously

•Usually there is a primary (dominant) disorder

•pH may be normal or near normal

•But the pH is usually outside compensatory limits of primary disorder

Examples of mixed acid-base disorders
Cardiac arrest, pulmonary oedema

pH 7.18

PCO2 6.7

HCO3 18
Mixed metabolic and respiratory acidosis
COPD and diuretic therapy

pH 7.42
PCO2 8.9
HCO3 42
Mixed respiratory acidosis and metabolic alkalosis
Salicylate poisoning

pH 7.39
PCO2 3.2
HCO3 14
Mixed respiratory alkalosis and metabolic acidosis
DKA with vomiting

pH 7.42

PCO2 5.3

HCO3 25

AG 23
Mixed metabolic acidosis and metabolic alkalosis

(In this case look out for high anion gap)

 Pokok Bahasan 5
Hb: Struktur, fungsi dari derivat dan
variannya
 Hemoglobin
synthesis, structure & function
Introduction

 The hemoglobin are red globular proteins which

have a molecular weight of about 64,500 and
comprise almost one third of the weight of a red
cell. Their primary function is the carriage of
oxygen from the lungs to the tissues.
 Over 500 different haemoglobin variants have
been described but all share the same basic
structure of four globin polypeptide chains each
with haem group. Functional haemoglobin
composed of two pairs of dissimilar globins.
Haemoglobin synthesis

Although haem & globin synthesis separately

within developing red cell precursors their
rate of synthesis are carefully coordinated to
ensure optimal efficiency of haemoglobin
assembly.
1st : Haem synthesis
The first step in
haem
synthesis is
the
combination of
succinyl CoA
and glycin to
produce δ
aminolaevulini
c acid (δ ALA).
This reaction is
energy
dependent and
so occurs in
the
mitochondria.
Haem synthesis
 It’s catalyzed by the enzyme δ ALA synthetase.
 This step is a first-limiting step for the whole process of
haem synthesis.
 It is stimulated by the presence of globin chains and
inhibited by the presence of free haem groups.
 This represents an important control mechanism of the rate
of haem synthesis and it’s coordination with globin
synthesis.
 Several factors are required for this step, including vitamin
B6, free ferrous and copper ions.
 Synthesis of the enzyme δ ALA synthetase is inhibited by
the presence of free haem.
 This represents a further feedback mechanism for haem
synthesis.
Mitochondrial δ -aminolevulinic acid (ALA) is transported
to the cytoplasm, where ALA dehydratase (also called
porphobilinogen synthase) dimerizes 2 molecules of
ALA to produce the pyrrole ring compound
porphobilinogen (PBG).
Haem synthesis
 The next step requires the synthesis of porphyrin
ring.
 The reactions involved are extremely complex
but can be summarized as the condensation of
four PBG molecules to form the asymmetric
cyclic uroporphyrinogen III (UPGIII).
 Synthesis of UPGIII requires the presence of two
enzymes (uroporphyrinogen I synthetase and
uroporphyrinogen III cosynthetase) and involves
the formation of several short-lived
intermediates.
 UPG III is converted to coproporphyrinogen III
(CPGIII) by decarboxylation of the acetate side
chains under the influence of the enzyme
uroporphyrinogen decarboxylase.
 CPGIII enters the mitochondria where it converted to
protoporphyrinogen IX (PPG IX) by an unknown
mechanism. This reaction is catalyzed by the enzyme
coproporhyrinogen oxidase.
PPG IX is further converted within the mitochondria to
protoporphrin IX.
 It only remains for the central ferrous ion to be inserted
to complete the synthesis of haem. This reaction is
catalyzed by the enzyme ferrochelatase and requires the
presence of reducing agents.
2nd : Globin synthesis
 Humans normally carry 8 functional globin
genes, arranged in two duplicate gene
clusters:
 The β-like cluster on the short arm of
chromosome 11.
 The α-like cluster on the short arm of
chromosome 16.
 These genes code for 6 different types of
globin chains: α,β,γ,δ,ε,ζ, globin.
Ontogeny of globin synthesis
Time Region Type of Globin Type of Hb
Gene

3 weeks of Yolk Sac ζ&ε Hb Gawer1 ζ ε)2

Gestation (

5 weeks of Yolk Sac γ&α Hb Portland(ζ γ)2

Gestation Hb GawerII (αε)2

6-30 weeksof Liver & spleen α & γ & β Hb F (α γ)2

Gestation

30 weeks of Liver δ Hb A2 (α δ)2

Gestation

At Birth B.M ___ HbA(α β)2

Adult haemoblobin
Hb A Hb A2 Hb F

structure a2b2 a2d2 a2g2

Normal % 96-98 % 1.5-3.2 % 0.5-0.8 %

2nd : Globin synthesis
Haemoglobin Structure
 Primary structure of globin

The primary structure of globin refers to the amino acid

sequence of the various chain types. Numbering
from the N-terminal end identifies the position of
individual amino acids. The identity and position of
these amino acids cannot be changed without
causing gross impairment to molecular function.
Secondary Structure of globin :

 The secondary structure of all globin chain types

comprises nine non-helical sections joined by
eight helical sections.

 The helical sections are identified by the letters

A-H while the non helical are identified by a pair
of letters corresponding to the adjacent helices
e.g. NA (N-terminal end to the start of A helix), AB
(joins the A helix to the B helix) etc.
Tertiary Structure of globin:

 The tertiary folding of each globin chain forms

an approximate sphere.

 The intra-molecular bonds which give rise to

the helical parts of the impart considerable
structure rigidity, causing chain folding to
occur in the non-helical parts.
 Tertiary folding gives rise to at least 3 functionally
important characteristics of the hemoglobin molecule :
1- Polar or charged side chains tend to be directed to the
outside surface of the subunit and, conversely, non-polar
structures tend to be directed inwards. The effect of this is to
make the surface of the molecule hydrophilic and the interior
hydrophobic.

2- An open-toped cleft in the surface of the subunit known as

haem pocket is created. This hydrophobic cleft protects
the ferrous ion from oxidation.

3- The amino acids, which form the inter-subunit bonds

responsible for maintaining the quaternary structure, and
thus the function, of the haemoglobin molecule are
brought into the correct orientation to permit these bonds
to form.
Quaternary structure of Haemoglobin

The quaternary structure of haemoglobin has four

subunits arranged tetrahedrally. In adult
haemoglobin- (HbA), there are different contact
areas:

 α1β1 and α2 β 2 which confirms stability of

the molecule.
 α1 β2 and α2 β1 which confirms solubility
of the molecule.
 α1 α2 and β1 β2 which are weak bonds to
permit oxygenation and deoxygenation.
Functions of Haemoglobin
 Oxygen delivery to the tissues
 Reaction of Hb & oxygen

 Oxygenation not oxidation

 One Hb can bind to four O2 molecules
 Less than .01 sec required for oxygenation
 b chain move closer when oxygenated
 When oxygenated 2,3-DPG is pushed out
 b chains are pulled apart when O2 is unloaded, permitting
entry of 2,3-DPG resulting in lower affinity of O2
Oxy & deoxyhaemoglobin
Normal Hemoglobin Function
 When fully saturated, each gram of hemoglobin binds
1.34 ml of oxygen.
 The degree of saturation is related to the oxygen tension
(pO2), which normally ranges from 100 mm Hg in arterial
blood to about 35 mm Hg in veins.
 The relation between oxygen tension and hemoglobin
oxygen saturation is described by the oxygen-
dissociation curve of hemoglobin.
 The characteristics of this curve are related in part to
properties of hemoglobin itself and in part to the
environment within the erythrocyte, including pH,
temperature, ionic strength, and concentration of
phosphorylated compounds, especially 2,3-
diphosphoglycerate (2,3-DPG).
Hb-oxygen dissociation curve
Normal Hemoglobin Function
 Oxygen affinity of hemoglobin is generally expressed in
terms of the oxygen tension at which 50% saturation
occurs.
 When measured in whole erythrocytes, this value
averages 27.1 mm Hg in normal, nonsmoking males and
27.5 mm Hg in normal, nonsmoking females.
 When oxygen affinity is increased, the dissociation
curve is shifted Leftward, and the value is reduced.
 Conversely, with decreased oxygen affinity, the curve is
shifted to the right.
Hb-oxygen dissociation curve
 The normal position of curve depends on

 Concentration of 2,3-DPG
 H+ ion concentration (pH)
 CO2 in red blood cells
 Structure of Hb
Hb-oxygen dissociation curve
 Right shift (easy oxygen delivery)

 High 2,3-DPG
 High H+
 High CO2
 HbS

 Left shift (give up oxygen less readily)

 Low 2,3-DPG
 HbF
Bohr Effect
 The change in oxygen affinity with pH is known
as the Bohr effect.
 Hemoglobin oxygen affinity is reduced as
the acidity increases.
 Since the tissues are relatively rich in carbon
dioxide, the pH is lower than in arterial blood;
therefore, the Bohr effect facilitates transfer of
oxygen.
 The Bohr effect is a manifestation of the acid-
base equilibrium of hemoglobin.
2,3-diphosphoglycerate
(2,3-DPG)
 This compound is synthesized from glycolytic intermediates by
means of a pathway known as the Rapoport-Luebering shunt.
 In the erythrocyte, 2-3-DPG constitutes the predominant
phosphorylated compound, accounting for about two thirds of the
red cell phosphorus.
 The proportion of 1,3-DPG pathway appears to be related largely to
cellular ADP and ATP levels; when ATP falls and ADP rises, a
greater proportion of 1,3-DPG is converted through the ATP-
producing step.
 This mechanism serves to assure a supply of ATP to meet cellular
needs.
 In the deoxygenated state, hemoglobin A can bind 2,3-DPG in a
molar ratio of 1:1, a reaction leading to reduced oxygen affinity
and improved oxygen delivery to tissues.
 When oxygen is unloaded by the hemoglobin
molecule and 2,3 DPG is bound, the molecule
undergoes a conformational change becoming what is
known as the ""Tense" or "T" form.
 The resultant molecule has a lower affinity for
oxygen.
 As the partial pressure of oxygen increases, the 2,3,
DPG is expelled, and the hemoglobin resumes its
original state, known as the "relaxed" or "R" form, this
form having a higher oxygen affinity.
 These conformational changes are known as
"respiratory movement".
 The increased oxygen affinity of fetal hemoglobin
appears to be related to its lessened ability to bind
2,3-DPG.
 The increased oxygen affinity of stored blood is
accounted for by reduced levels of 2,3-DPG.
 Changes in 2,3-DPG levels play an important
role in adaptation to hypoxia. In a number of
situations associated with hypoxemia, 2,3-DPG
levels in red cells increase, oxygen affinity is
reduced, and delivery of oxygen to tissues is
facilitated.
 Such situations include abrupt exposure to high
altitude, anoxia due to pulmonary or cardiac
disease, blood loss, and anemia.
 Increased 2,3-DPG also plays a role in
adaptation to exercise. However, the compound
is not essential to life; an individual who lacked
the enzymes necessary for 2,3-DPG synthesis
was perfectly well except for mild polycythemia
Carbon Dioxide
(CO2)
 Transport of carbon dioxide by red cells,
unlike that of oxygen, does not occur by
direct binding to heme.
 In aqueous solutions, carbon dioxide
undergoes a pair of reactions:
1. CO2 + H2O H2CO3

2. H2CO3 H+ + HCO3
(CO2)
 Carbon dioxide diffuses freely into the red cell where the
presence of the enzyme carbonic anhydrase facilitates
reaction 1.
 The H+ liberated in reaction 2 is accepted by deoxygenated
hemoglobin, a process facilitated by the Bohr effect.
 The bicarbonate formed in this sequence of reactions diffuses
freely across the red cell membrane and a portion is
exchanged with plasma Cl-, a phenomenon called the
"chloride shift." the bicarbonate is carried in plasma to the
lungs where ventilation keeps the pCO2 low, resulting in
reversal of the above reactions and excretion of CO2 in the
expired air.
 About 70% of tissue carbon dioxide is processed in this way.
Of the remaining 30%, 5% is carried in simple solution and
25% is bound to the N-terminal amino groups of
deoxygenated hemoglobin, forming carbaminohemoglobin.
Methemoglobinemia
 In order to bind oxygen reversibly, the iron in the
heme moiety of hemoglobin must be maintained
in the reduced (ferrous) state despite exposure
to a variety of endogenous and exogenous
oxidizing agents.

 The red cell maintains several metabolic

pathways to prevent the action of these oxidizing
agents and to reduce the hemoglobin iron if it
becomes oxidized. Under certain
circumstances, these mechanisms fail and
hemoglobin becomes nonfunctional.
Methemoglobinemia
 At times, hemolytic anemia supervenes as
well. These abnormalities are particularly
likely to occur
(1) if the red cell is exposed to certain oxidant drugs
or toxins
(2) if the intrinsic protective mechanisms of the cell
are defective or
(3) if there are genetic abnormalities of the
hemoglobin molecule affecting globin stability or
the heme crevice.
 Pokok Bahasan 6
Gangguan oksigenasi jaringan
Respiratory failure is a severe clinical
endpoint of pulmonary disease
Respiratory failure can be caused by

Acute pulmonary disease

-Pneumonia
-Pulmonary oedema
-ARDS
-Acute asthma
-Pulmonary embolism
-Atelectasis (collapse)

Chronic pulmonary disease

-COPD (Chronic Bronchitis/Emphysema)
-Pulmonary fibrosis
 How is respiratory failure classified?

Type 1 Respiratory Failure

Hypoxaemia (normo or hypocapnia)

failure of O2 transfer

• Ventilation-perfusion (V/Q) defects

• Right-to-Left shunts e.g. pulmonary oedema

Type 2 Respiratory Failure

Hypoxaemia and Hypercapnia

failure of ventilation to remove CO2

• Reduced total ventilation

• Decreased diffusion
 Pokok Bahasan 7
Dasar Biokimia Analisa Gas Darah
Arterial Blood Gas (ABG) analysis is an
essential investigation for definitive diagnosis
of respiratory failure
 How is Respiratory failure defined using ABG?

Type 1 Respiratory Failure (low PO2, normal/low PCO2)

-PO2 < 8.0 kPa

-PCO2 < 6.7 kPa

Type 2 Respiratory Failure (low PO2, high PCO2)

-PO2 < 8.0 kPa

-PCO2 > 6.7 kPa

 Use of Biochemistry Tests in Pleural Fluid analysis

Main purpose is to differentiate Transudative and Exudative effusions

Transudates
•CCF
•Cirrhosis
•Nephrotic syndrome

Exudates
•Malignancy
•Infection e.g. bacterial pnemonia, TB
•PE
•GI disease e.g pancreatitis
•Chylothorax
•Connective tissue disorders
Use of Biochemistry Tests in Pleural Fluid analysis (2)

Traditionally pleural fluid protein level

• < 30g/L in Transudate
• >30g/L in Exudate
• Misclassification in 10%

Light’s Criteria for Exudative Pleural effusion

Any one of the following

•Pleural fluid :Plasma Protein ratio >0.5

•Pleural fluid:Plasma LDH >0.6
•Pleural fluid > 2/3 upper limit of normal plasma LDH
Other Biochemical Tests used in Pleural Fluid Analysis

Amylase e.g. pancreatic disease, malignancy

Glucose <3.4 mmol/L

-Malignancy
-TB
-Empyema
-Rhematoid arthritis

Chylothorax

The following criteria apply

•Pleural fluid triglyceride >1.25 mmol/L
•Pleural fluid:Plasma Triglyceride >1.0
•Pleural fluid:plasma cholesterol <1.0
Miscellaneous Biochemistry Tests in Pulmonary Disease

Serum Angiotensin Converting enzyme (ACE)

-Increased in 75% patinet with sarcoidosis
-Reflects activity of disease
-?Use in monitoring response to treatment

α1-antitrypsin deficiency
-Multiple phenotypes (genotypes)
-Associated with susceptibility to emphysema

Cystic Fibrosis
-Genotyping e.g. Δ508
-Diagnosis Sweat Test
-Screening using immunoreactive trypsin (IRT)
Biochemistry of Lungs
• Produce: surfactant
collagen + elastin
mucus (mucopolysacharides + IgA)
• Activate: angiotensin (ACE in luminal surface of the
pulmonary endothelium)

• Inactivate: ROS
kinins (hydrolysis of peptide bonds in bradykinin)
serotonin (from mast cells, from blood - MAO)
acetylcholin
detoxication of foreign components
(cytochrom P450 in microsomes)lbiochemistry of
lungs
 Pulmonary Surfactant
• surface-active lipoprotein complex formed by type II alveolar cells
• complex of proteins and lipids with a hydrophilic and a
hydrophobic region
• the hydrophilic head groups facing towards the water and the
hydrophobic tails facing towards the air
• reduces surface tension
• surface tension is an effect within the surface layer of a liquid that
causes that layer to behave as an elastic sheet
• increases pulmonary complience (the ability of the lungs to stretch in a
change in volume relative to an applied change in pressure).
Properties of Surfactant
• Once secreted to alveolar space, surfactant absorbs
rapidly to the air-liquid interface (a newborn baby´s
first breath).

• Once in the interface, surfactant films reduces

surface tension when compressed during expiration
(lungs don´t collapse).

• Surfactant proteins recognize and opsonize

bacterial, fungal, viral surface oligosaccharides.
Lyra, P.P.R.; de Albuquerque Diniz, E.M. Clinics 62: 181, 2007
Structure of Alveolus

http://herkules.oulu.fi/isbn9514270584/html/c273.html
Phospholipides

Nonpolar tail Polar head

Proteins
Synthesis - epitelial cells

SP-A and SP-D

 large glykosylated proteins ( SP-D
has 355 AA)
 water-soluble
 members of calcium-dependent
carbohydrate-binding collectin family

SP-B and SP-C

 small peptides (35 AA), highly
hydrophobic
 confer surface tension-lowering
properties
 important for spreading of the
surfactant
Proteins

SP-A is responsible for:

• formation of tubular myelin
• regulation of phospholipid insertion into the monolayer
• modulation of uptake and secretion of phospholipids by type II cells
• activation of alveolar macrophages
• binding and clearance of bacteria and viruses
• chemotactic stimulation of alveolar macrophages
SP-D plays important role in pathogen defence
SP-B and SP-C:
• enhance the biophysical properties of surfactant
• assist in rapid insertion of phospholipids into the monolayer and
molecular ordering of the monolayer
Surfactant Metabolism
 DPPC is synthesized
in rER.

 Transferred to the
lamellar bodies together
with SP-B and SP-C (the
lamellar bodies are the
storage and secreting
granules surrounded by
a limiting membrane that
fuses with the plasma
membrane).

 Surfactant secretion
can be stimulated by the
stretching of the type II
epithelial cells, by the
action of beta-agonists,
and purinergic agonists,
such as ATP

Lyra,P.P. R.; de Albuquerque Diniz, E.M. Clinics 62: 181, 2007

• Lamellar bodies and tubular myelin
• Lamellar bodies have an acidic internal environment and have high
calcium content.
Control of Surfactant Release
Distortion of cells
Hyperventilation - deep breath, yawn
acetylcholine (large doses)
beta-agonists
purinoreceptors
corticoids (maturity after pre-term birth)
thyroxin
Synthesis

glycerol glucose from circulation (glykogen)

polar heads cholin, inositol from circulation
fatty acids endogenous from lactate
exogenous
Synthesis of DPPC de novo
Glucose Glycogen

NAD+ NADH
glycerol-3-phosphate DHAP
palmitoyl-CoA

CoASH
palmitoyl-G3P Choline
palmitoyl-CoA ATP

CoASH ADP
dipalmitoylphosphatidic acid phosphocholine
H2O CTP

Pi PPi
dipalmitoylglycerol CDP-choline
CMP
DPPC
 Function of Surfactant

• surfactant reduces tension of the air/liquid interface to

near zero
• surfactant aids the oxygen to perfuse from the
atmosphere into the pulmonary capillaries

During the breathing cycle :

• DPPC is insert into the monolayer, lowers the
surface tension of air/liquid interface
• PG is effective in spreading surfactant
• Proteins accelerate the process to lower tension
Reactive Oxygen Species -
ROS
.
O - + e + H+ HO
. hydroperoxid radical
2 2

.
HO2 H+
.
+O - superoxid radical
2

. hydrogen peroxide
O2- + 2H+ + e H2 O2

H2O2 + e OH- + OH
. hydroxyl radical

.OH + e + H+ H2 O
Reactive Oxygen Species -
ROS
O2 + 2H2 2H2O

Cu,Zn-SOD
. .
O2 e O2 - e 2H H2O2 e H OH e H H2O
Fe3+
+ MPO Px
Cu
.
OH H 2O
HClO
peroxidation of lipids (phospholipids) aldehydes (malonaldehyde)
O3, NO, NOx, SiO2, smoking, infection, radiation, hypoxia/reoxygenation,
ischemia/reperfusion
Reactive Nitrogen Species - RNS
L-arginine
NOS
L-citrulline + NO
HbO2 O2

nitrate
O2- nitrite

NO3- + metHb peroxinitrite NO2- HOCl + MPO

ONOOH
oxidation nitration
nitrosation

thiyl radical S-nitrosothiol nitrotyrosine

Antioxidant Defence
Free radical scavenging enzymes

Components of antioxidant protection

Enzymatic Antioxidative Defence
tetravalent reduction (ROS generated during oxidative phosphorylation)

acceleration of monovalent reduction:

SOD cytosolic (Cu-Zn) 5
mitochondrial (Mn) 1
extracellular (Cu-Zn)
catalase (heme-containing)
glutathione system GPx (Se)
2 cytosolic, membrane-ass., extracellular
2H2O 2GSH NADPH Rib-6-P

GPx GR
reductase
H2O2 GSSG NADP+ Glucose
Nonenzymatic Scavengers

vitamin E - lipid peroxyl radicals

vitamin C - O2-, .OH , Fe3+ Fe2+
β-carotene (O2-), uric acid (O2-), glucose (OH),
bilirubin (LOO.)
Fe sekvestration – lactoferrin and transferrin – ferric ions
ceruloplasmin utilize H2O2 for reoxidation of copper

Lung: intracellular enzymes

epithelial lining fluid (GSH 100x higher than in
plasma, catalase, SOD, GPx)
Collagen

90% I and III, type II, V, VIII

Synthesis Degradation
Deposition

Fibrosis Emphysema

Degradation – specific MMP´s

TIMP´s – tissue inhibitors of MMP´s