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Alanine transaminase (ALT/GPT) catalyzes the reaction

These two enzymes are abundant in tissues, particularly liver and heart tissue.
Quantifying these two enzymes in serum is clinically significant, especially in the diagnosis,
treatment, and monitoring of liver and heart diseases.
AST and ALT are transaminases that are abundant in tissues such as the heart muscle,
skeletal muscle, and liver. In liver and heart diseases, determining the activity of these
enzymes is very valuable.
- In myocardial infarction, these enzymes escape from muscle cells due to membrane
damage and spill into the blood. Quantifying AST and ALT in serum can provide information
about myocardial damage, especially in acute damage.
- AST and ALT are also important for identifying liver damage diseases such as viral
hepatitis, tetrachloride carbon poisoning, chloroform poisoning, or poisoning from solvents
used in laundry technology. These toxins cause liver damage, leading to the release of
enzymes into the serum.
[Link]. Aminodepletion by aminodepletion
There are various types of deamination reactions in the body, such as hydrolytic
deamination, intramolecular deamination, and oxidative deamination, but the most important
and common is oxidative deamination.
The oxidative deamination process occurs in two steps:
 + Oxidation of amino acids into imine acids
+ Spontaneous hydrolysis of imine acid to form α-keto acid and NH3.

- Mechanism:

- Characteristics:
+ The coenzyme FADH2 does not enter the cellular respiratory chain but is directly
transferred to oxygen to form H₂O₂ (toxic). Subsequently, H₂O₂ is immediately broken down into
H₂O and O₂(non-toxic) by the enzyme catalase, which is also present in the endoplasmic
reticulum. This is the detoxification mechanism that frequently occurs in the body.
+ The catalytic enzyme amino oxidase is present in the endoplasmic reticulum but has
low activity, so it plays a minor role in the deamination of amino acids. However, with
glutamic acid, the oxidative deamination reaction occurs easily.
The oxidative deamination reaction of glutamic acid
Glutamic acid (primarily formed through amino acid exchange) undergoes oxidative
deamination under the action of glutamate dehydrogenase according to the following reaction:

- Characteristics:
+ The coenzyme is NADH H+(NADPH H+) formed and enters the cellular respiratory
chain. Therefore, this process is non-toxic.
+ The enzyme glutamate dehydrogenase has high activity. Therefore, the oxidative
deamination of glutamic acid plays a crucial role.
 + Glutamate dehydrogenase is regulated by the concentration of ADP (stimulation) and
GTP (inhibition), which are products formed in the citric acid cycle.
[Link]. Relationship between the two processes of exchange and deamination
Amino oxidases have low activity, so the oxidative deamination of most amino acids is
difficult to carry out, except for glutamic acid, which is easily deaminated because glutamate
dehydrogenase has high activity. On the other hand, all transaminases have high activity, so
the amino acid exchange reaction is easily carried out.
Therefore, amino acids that want to remove the amino group must go through two steps:
- Step 1: Exchange the amino group with α-ketoglutaric acid to form glutamic acid, as
transaminase has strong activity.
- Step 2: Glutamic acid undergoes oxidative deamination, as glutamate dehydrogenase
has strong activity.
As a result, all amino acids lose their amino groups.

Figure 10.3. Relationship between the two processes of exchange and deamination

2.1.2. Removal of the carboxyl group (-CO2)

In animal tissues, some amino acids undergo CO₂ removal to form the corresponding
amines via decarboxylase enzymes. Most of these amines have high biological roles; after
fulfilling their biological functions, they undergo further degradation.
 The deaminated products of certain amino acids have high biological significance:

Histamine is a substance that dilates blood vessels, contracts smooth muscles, and
increases vascular permeability.

Gamma-aminobutyric acid (GABA) is a substance that regulates nervous system

activity and inhibits nerve reflexes.
Typically, after these amines lose their effect, they continue to undergo oxidative
deamination by monoamine oxidase or diamine oxidase, producing NH3 and aldehydes. The
aldehydes are further oxidized into carboxylic acids, which then degrade into acetylCoA and
enter the Krebs cycle to produce CO2, H2O, and ATP. This demonstrates the energy-producing
role of amino acids.
Specifically:

2.2. The fate of ammonia (NH₃)

2.2.1. The synthesis and transport of glutamine
NH3 is primarily formed in tissues through the process of deamination, with a smaller
portion resulting from the degradation of nitrogen bases in nucleic acids. NH3 is toxic to the
body. In the blood, NH3 is transported in the form of glutamine, which is formed by combining
with glutamic acid.
 (non-toxic) with the help of the enzyme glutamine synthetase and ATP providing energy. Therefore
the concentration of free NH3 in the blood is very low (0.1–0.2 mg/L).
Glutamine is transported via the blood to the liver and kidneys. Here, glutamine is
hydrolyzed into NH3 and glutamic acid by the enzyme glutaminase.
- In the liver: NH3 is used by the liver to synthesize urea and is primarily excreted
through the kidneys – urine via filtration, and partially through feces, sweat, and breath. In
birds and reptiles, NH3 is synthesized into uric acid and excreted. In many aquatic animals such
as fish, NH3 is directly released into the environment.
- In the kidneys: in the renal tubule cells, NH3 combines with H+to form NH4(+), whichis then excreted
excreted in urine, thereby contributing to acid-base balance regulation.

Tiss
ue

Bloo
d

Kidne Live
y r

Urine

Figure 10.4. The synthesis and transport of glutamine in the body

Additionally, NH3 can be utilized by the body for the resynthesis of amino acids or the
synthesis of nucleic acids.
In the brain, glutamine synthesis is the primary pathway for removing NH3, preventing toxicity to
the
brain. The brain also has the ability to synthesize urea, but this is insignificant, so it does not play
an
in NH3 removal. NH3 levels in cerebral blood are often high, but the glutamate supplied by the
blood to the brain is insufficient for glutamine synthesis, so the brain must synthesize
glutamate itself to meet this demand. The direct substrate for glutamate synthesis in the brain
is α-ketoglutarate, an intermediate product of the citric acid cycle.
2.2.2. Urea synthesis (urea cycle or ornithine cycle)
Occurs primarily in the liver through five reactions:
- Formation of carbamoyl phosphate (mitochondrial)
Produced from NH3 + CO2, the enzyme is carbamoyl phosphate synthetase.

- Forms citrulline (mitochondria)

Using carbamoyl phosphate + ornithine, the enzyme is ornithine carbamoyltransferase
(OCT).

After being synthesized in the mitochondria, citrulline leaves the mitochondria into the
cytoplasm and continues to participate in subsequent reactions.
- Formation of arginosuccinate (cytoplasm)
Using citrulline + aspartic acid, the enzyme is arginosuccinate synthetase.
 - Cleavage of the arginosuccinate bond to form arginine
With the enzyme arginosuccinase.

- Hydrolyzing arginine to form urea

The enzyme catalyzing this reaction is arginase.

Thus, through five reactions, a urea molecule has been synthesized from NH3 and ornithine
is regenerated and re-enters the next urea cycle.
 The synthesis of urea by the liver is considered an endogenous detoxification function
that occurs regularly in the body. Blood urea levels depend on diet, increasing slightly in a
high-protein diet, and increasing pathologically in conditions causing kidney failure, increased
metabolism (cancer, prolonged high fever, diabetes, etc.). Decreased blood urea is seen in
end-stage liver failure (often accompanied by increased blood NH3). In end-stage liver failure
(decompensated liver failure) such as end-stage liver cancer, cirrhosis, or end-stage chronic
hepatitis, patients may experience coma due to elevated blood ammonia levels accompanied
by foul-smelling breath (hepatic encephalopathy).

Mitoch
ondria

(Ornithine transport
system)

Figure 10.5. The urea cycle and its relationship with the Krebs cycle

Sources of substances that make up urea

molecules:

Free CO2 Free NH3 supply

Asp provided

The urea cycle is linked to the Krebs cycle through two main products: oxaloacetic acid and
fumaric acid.
2.3. The fate of the carbon skeleton of amino acids (α-keto acids)
After losing the amino group, the remaining carbon skeleton of amino acids (α-keto
acids) continues to degrade into various intermediate products. Most of these products belong
to the Krebs cycle, such as: pyruvate, oxaloacetate, α-ketoglutarate, succinylCoA,
acetoacetylCoA, acetylCoA, etc.
From these intermediates, they will continue to be metabolized through different pathways
pathways, which may include the following:
- Undergo complete degradation through the Krebs cycle, producing CO2, water, and ATP.
- Glucose synthesis (role of amino acids in sugar production).
- Fatty acid synthesis (role of amino acids in ketone body formation).
- Or the resynthesis of amino acids.

Figure 10.6. Fate of the C-skeleton of amino acids

3. AMINO ACID SYNTHESIS

Amino acids are the building blocks of all proteins in the body. Different organisms
have varying capabilities for amino acid synthesis:
 - Higher plants: capable of synthesizing all amino acids from NH₃, nitric acid, or nitrate.
Leguminous plants can utilize atmospheric nitrogen to synthesize amino acids.
- Microorganisms: depending on the type, they can also use the above raw materials to
synthesize all types of amino acids. However, there are also types that cannot synthesize them
themselves and must obtain them from the environment.
- Humans and higher animals: They cannot synthesize all amino acids but only some,
with the remainder obtained from dietary proteins.
Based on these capabilities, amino acids are classified into the following types:
* Essential amino acids: These are amino acids that the bodies of humans and higher
animals cannot synthesize on their own. Without them, the body cannot develop properly, and
they must be obtained from dietary protein sources.
* Non-essential amino acids: These are amino acids that humans and higher animals
can synthesize.
* Semi-essential amino acids: These are amino acids that the human body and higher
animals can synthesize in small quantities under specific physiological conditions, such as in
children, young animals, after illness, or in the elderly.

Table 10.1. Essential and non-essential amino acids

Essential amino acids Non-essential amino acids
Lysine Glycine
Threonine Alanine
Methionine Cysteine
Valine Glutamic acid
Leucine Glutamine
Isoleucine Aspartic acid
Phenylalanine Tyrosine
Arginine (semi-essential) Glycine

Histidine (semi-essential) Alanine

For non-essential amino acids, each amino acid is synthesized through distinct, complex
pathways using specific enzymes. However, there are some common pathways involved in
their synthesis.
3.1. Some common pathways for amino acid synthesis.
- Synthesis by aminating the corresponding α-keto acid: the synthesis of glutamic
acid.
 This reaction is of particular importance for the synthesis of amino acids in all
organisms, as glutamate plays a major role in the deamination of amino acids.
From glutamic acid, glutamine can be synthesized.

This is also an important reaction. In the body, glutamine can be used to synthesize
many biologically important substances such as nitrogenous bases, glucosamine, etc.
- Synthesis via amino acid exchange reactions: the synthesis of alanine and aspartic acid.

Aspartic acid can be aminated to synthesize asparagine.

- Synthesis of nonessential amino acids from essential amino acids: this method
is typically performed with amino acids that have similar structures. For example:
From phenylalanine to tyrosine: tyrosine is formed by the hydroxylation of
phenylalanine, catalyzed by the enzyme phenylalanine hydroxylase. This reaction is
irreversible, so tyrosine cannot replace alanine in the diet.
From glycine to serine.
From methionine to cysteine: cysteine is formed through the degradation of methionine.
3.2. Amino acids are precursors to many other compounds
In addition to being the raw materials for protein synthesis, amino acids are also
precursors for many biosynthetic processes of other substances, especially those with
important biological functions in the body.
3.2.1. Porphyrin synthesis
The porphyrin ring is synthesized from glycine and succinylCoA. The porphyrin
molecule contains 4 nitrogen atoms and 8 carbon atoms provided by glycine.
3.2.2. Purin and pyrimidine synthesis
Glycine, aspartate, and glutamine are amino acids used by the body to synthesize
purine nitrogen bases.
Aspartate is required for the synthesis of pyrimidine nitrogen bases.
Purin and pyrimidine bases are structural components of coenzymes belonging to the
nucleotide derivative group, such as NAD+ , FAD…
3.2.3. Hormone synthesis
Hormones are derivatives of amino acids, such as thyroid hormones: thyroxine (T4),
triiodothyronine (T3), or adrenal hormones: adrenaline and noradrenaline. These hormones
are synthesized from tyrosine and phenylalanine.
3.2.4. Synthesis of nicotinic acid
The human body can synthesize nicotinic acid (vitamin PP) from tryptophan. It has been
observed that a diet lacking in tryptophan-rich proteins can also cause symptoms similar to
vitamin PP deficiency.
3.2.5. Synthesis of creatine, creatine phosphate, and creatinine
There are three amino acids involved in creatine synthesis: arginine, glycine, and
methionine. Methionine in its activated form (adenosine methionine) is a supplier of the -CH3
group for many reactions. In muscles, the brain, and blood, creatine exists in two forms: free
creatine and creatine phosphate, with creatine phosphate being the important energy storage
form in muscles.
The formation of creatine, creatine phosphate, and creatinine is
as follows: Creatine is synthesized from three amino acids: Gly,
Arg, Met.

Creatine kinase catalyzes the transfer of the phosphate group from ATP to creatine,
forming creatine phosphate when the muscle is at rest, and regenerating ATP when needed.
Dehydrated creatine forms a cycle
to form creatinine. Each day, a constant amount of creatinine is produced, which is excreted
by the kidneys into the urine. The amount of creatine and creatinine is proportional to muscle
mass.

3.2.6. Synthesis of glutathione

Glutathione is a tripeptide composed of three amino acids: glutamic acid, cysteine, and
glycine. Glutathione exists in two forms: oxidized and reduced. Due to the –SH group of
cysteine, glutathione can donate or accept hydrogen.
Glutathione is a coenzyme for many oxidation-reduction enzymes. Additionally,
glutathione protects membrane lipids by counteracting lipid oxidation.

4. AMINO ACID DISORDERS

Amino acid disorders (aminoacidopathies) caused by amino acid metabolism disorders
are rare genetic disorders. Abnormalities occur either due to a deficiency of specific enzymes
in the metabolic pathway or abnormalities in the amino acid transport system across the
membrane. These disorders increase the amount of amino acids in the blood, increase the
excretion of free amino acids in the urine, along with clinical signs corresponding to the
disorder.
DISCUSSION CONTENT
1. Common degradation pathways of amino acids. The process of amino acid elimination
through amino acid exchange and oxidative deamination, and the relationship between
these two processes.
2. The fate of ammonia (NH3).
- The synthesis and transport of glutamine.
- The synthesis of urea.
3. The decarboxylation process of amino acids.
 Chapter 11
HEMOGLOBIN METABOLISM
OBJECTIV
ES
After completing this chapter, students will be able to:
1. Describe the degradation process of hemoglobin.

2. Analyze hemoglobin metabolism disorders and their clinical applications.

Hemoglobin is a mixed protein belonging to the chromoprotein group, with the outer
group being HEM found in red blood cells that perform the task of transporting gas.
In terms of structure, Hb consists of two parts:
- Globin: is the pure protein component, consisting of four polypeptide chains, two of
which are identical pairs, denoted as 2α and 2β. The globin component determines the
species-specific nature of hemoglobin (Hb).
- HEM is the heme group, composed of a porphyrin ring (protoporphyrin) bound to Fe2+.
HEM is identical across species.
In its structure, each polypeptide chain of globin binds one HEM. Therefore, a
hemoglobin molecule contains four HEM moieties.

Figure 11.1. Structure of hemoglobin

1. SYNTHESIS OF HEMOGLOBIN
Hemoglobin synthesis in adults occurs in immature red blood cells of bone marrow
cells, consisting of two processes: HEM synthesis and globin synthesis.
1.1. Hem Synthesis
A HEM molecule consists of a protoporphyrin (porphyrin) ring combined with Fe2+. All
tissues containing hemoproteins have the ability to synthesize HEM, but synthesis occurs
most strongly in immature red blood cells of bone marrow cells.
The mechanism of porphyrin synthesis in animal tissues has been elucidated using
radioactive isotopes (David Shemin).
Porphyrin biosynthesis plays a particularly important role in animals because the
porphyrin ring is an important structural component of hemoglobin, myoglobin, and many
porphyrin-containing enzymes such as cytochromes, catalase, peroxidase, etc. Additionally,
porphyrin is also an important structural component of chlorophyll in plants.
The raw materials for HEM synthesis are glycine and succinylCoA. The synthesis
occurs in five steps:

- Step 1: Formation of δ-aminolevulinic acid (ALA): occurs in the mitochondria.

From glycine and succinylCoA, CO2 and HSCoA are removed, with the enzyme δ-
aminolevulinic acid synthetase acting as the catalyst.
δ-aminolevulinic acid is transported from the mitochondria to the cytoplasm after synthesis.
- Step 2: Formation of porphobilinogen.
Two ALA molecules combine, releasing one molecule of H2O, catalyzed by the enzyme
ALA-dehydratase.
- Step 3: Formation of uroporphyrinogen (I, II, III).
- Step 4: Formation of coproporphyrinogen III and protoporphyrin III (Porphyrin).
After coproporphyrinogen III is formed by the carboxylase reaction of the acetate (A)
groups of uroporphyrinogen III, coproporphyrinogen III leaves the cytoplasm and enters the
mitochondria. Here, it undergoes decarboxylation and oxidation to form protoporphyrinogen
III.
Protoporphyrinogen III continues to oxidize at the methyl bridges to form
protoporphyrin (porphyrin).
- Step 5: Porphyrin binds Fe to form HEM.
In the mitochondria, porphyrin binds with Fe2+to form HEM, and plasma transferrin
transports iron to the hematopoietic organs.
After HEM is synthesized, it exits the mitochondria into the cytoplasm, where it binds
to globin to form hemoglobin.
 HEM α + HEM β form a dimer; two dimers combine to form a tetramer (Hb).
The enzyme catalyzing the ring-closing reaction to form porphobilinogen is inhibited by lead salts.
(Pb).
1.2. Globin synthesis and the combination of HEM and globin
Globin synthesis occurs in the cytoplasm of bone marrow cells via the protein
biosynthetic pathway.
After HEM is synthesized, it leaves the mitochondria into the cytoplasm, where it will
combines with globin to form Hb.
The combination of HEM and globin is achieved through a coordination bond between
the iron of HEM and the nitrogen of the imidazole ring (histidine) of globin.
The process of hemoglobin synthesis primarily occurs in hematopoietic organs such as
bone marrow and immature red blood cells. Immature red blood cells contain all the enzymes
necessary for the complete synthesis of hemoglobin molecules.
1.3. Factors affecting HEM synthesis
- HEM is a negative allosteric regulator of the enzyme δ-aminolevulinic acid synthase.
- Pantothenic acid, vitamin B12, and vitamin B6 are essential for HEM synthesis.
- Plasma transferrin transports iron to the hematopoietic organs.

2. HEMOGLOBIN DEGRADATION
The average lifespan of a red blood cell is 120 days, during which hemoglobin remains
unchanged. When red blood cells die (in the spleen), Hb is released and degraded.
The degradation of Hb produces the following products:
- Iron: mostly reused, the remainder is excreted (in feces).
- Porphyrin: undergoes further degradation to form bile pigments and is excreted
excreted (in feces).
- Globin: hydrolyzed to form amino acids and undergoes further degradation along the
amino acid pathway.
The degradation process consists of 5 stages:
- Stage 1: porphyrin ring opening by oxidation at Cα, CO2 elimination, cleavage of the
bond between pyrrole rings I and II to form verdoglobin.
 - Stage 2: The iron and globin of verdoglobin form biliverdin (green).

Iron: either reused or excreted (in feces).

Globin: hydrolyzed into amino acids and degraded via the amino acid pathway.
- Stage 3: Reduction of biliverdin to form free bilirubin (yellow).
Characteristics of free bilirubin:
+ Toxic.
+ Insoluble in water, soluble in lipids and yellow in color.
+ Because the diazo reaction is slow, it is also called indirect bilirubin.

The entire process occurs in the spleen.

- Stage 4: formation of conjugated bilirubin.
Free bilirubin, after being formed in the spleen, is transported in the blood bound to
serum albumin to the liver due to its toxic nature and insolubility in water.
In the liver: free bilirubin is conjugated with glucuronic acid by the liver to form
conjugated bilirubin (non-toxic) via the enzyme glucuronyl transferase. The formation of
conjugated bilirubin in the liver is an endogenous detoxification process that occurs
continuously in the body.
 Characteristics of conjugated bilirubin:
+ Non-toxic.
+ Water-soluble, yellow in color.
+ Reacts quickly with the diazo reaction, hence it is also called direct bilirubin.
- Stage 5: Liver-Intestine Cycle.
After being synthesized by liver cells, conjugated bilirubin is transported via the bile
ducts to the gallbladder for storage, then flows through the common bile duct into the
intestines to participate in the digestion of lipid-containing foods. Conjugated bilirubin is the
primary bile pigment in humans.
In the intestine: conjugated bilirubin is hydrolyzed into free bilirubin and glucuronic
acid by the enzyme glucuronidase. Glucuronic acid is reused, while free bilirubin is further
reduced by enzymes present in the gut's beneficial bacteria into two products: urobilinogen
and stercobilinogen.
The urobilinogen and stercobilinogen products in the small intestine are reabsorbed into
the blood (portal vein system) and returned to the liver for resynthesis into conjugated
bilirubin, with a small amount excreted in the urine. The majority remaining in the intestine is
excreted in the feces as urobilinogen and stercobilinogen.

Figure 11.2. Diagram of hemoglobin degradation (liver-intestine cycle)

 In feces and urine, urobilinogen and stercobilinogen products, after being excreted, react
with oxygen in the air to form urobilin and stercobilin (yellow), which give color to feces and
urine.
In cases of reduced intestinal bacteria (in dysbiosis due to prolonged antibiotic use),
bilirubin is not reduced and is excreted intact in the feces. Upon contact with air, bilirubin
auto-oxidizes to biliverdin, causing the feces to turn green. Similarly, in newborns during the
first few days after birth, due to a lack of intestinal bacterial enzymes, the feces also turn
green.

3. HEMOGLOBIN METABOLISM DISORDER

3.1. Hemoglobin Synthesis Disorders
3.1.1. Hem Synthesis Disorders
Genetic or acquired enzyme deficiency causes accumulation of porphyrin (HEM
precursor) metabolites in the bone marrow or liver, known as porphyrias. The disease
corresponds to enzyme deficiency in each step of HEM synthesis except ALA synthetase.
Porphyrias due to HEM synthesis disorders increase intermediate products in the blood, urine,
and feces.
3.1.2. Globin synthesis disorders
Globin is synthesized in the cytoplasm of bone marrow cells via the protein biosynthetic
pathway. All disorders related to globin are genetic in nature. Common disorders may result
from abnormalities in the β or γ chains, or an imbalance in their ratio, leading to
hemoglobinopathies (Chapter 3, Hemoglobin Chemistry section).
3.2. Hemoglobin degradation disorders
Normally, total serum bilirubin levels are < 1 mg/100 ml of blood (0.2
- 0.8 mg), mainly in the free form, with only trace amounts in the conjugated form. In the
blood, free bilirubin is transported in a protein-bound form (mainly albumin).
In pathological conditions, serum bilirubin levels may increase. If conjugated bilirubin
increases, due to its water-soluble nature, it can diffuse through blood vessel walls into tissues
(particularly the skin and mucous membranes), causing clinical jaundice. If unconjugated
bilirubin increases, it will separate from the blood vessel walls and diffuse into tissues
(especially fatty tissue), also causing bilirubin deposition in tissues (mainly the skin and
mucous membranes), resulting in jaundice and yellowing of the eyes.
Thus, for some reason, increased blood bilirubin (both free and conjugated) causes
jaundice (yellowing of the skin and eyes) in patients clinically.
Jaundice is classified into three main categories based on its cause:
3.2.1. Jaundice due to prehepatic causes (hemolysis)
Causes: include all cases of hemolysis, leading to massive red blood cell rupture.
+ Physiological: hemolytic jaundice in the neonatal period.
+ Pathology: malaria, blood transfusion errors, hemolytic anemia, snake bites, etc.
Due to hemolysis, Hb is released in large amounts, causing an increase in free bilirubin.
The liver increases the synthesis of free bilirubin into conjugated bilirubin, but because the
liver's ability to conjugate with glucuronic acid is limited, the increase in conjugated bilirubin
is insignificant.
Result: elevated free bilirubin, mildly increased conjugated bilirubin, leading to
increased total bilirubin. The patient presents with jaundice, icterus, and urine may contain
mild bile pigments but no bile salts.
3.2.2. Jaundice due to hepatic causes
Example: In cases of hepatitis, particularly viral hepatitis.
In this case, Hb metabolism in the spleen is normal. However, because liver
parenchymal cells are damaged, liver function is reduced, including the function of
synthesizing unconjugated bilirubin into conjugated bilirubin, leading to high levels of
unconjugated bilirubin in the blood. On the other hand, hepatitis also causes many liver cells
to rupture, while other cells become edematous, increasing membrane permeability and
compressing the bile ducts in the liver, obstructing bile flow to the gallbladder. Conjugated
bilirubin cannot flow to the gallbladder and then to the intestines, so it spills into the blood,
leading to an increase in both free and conjugated bilirubin in the blood.
Result: elevated free bilirubin, elevated conjugated bilirubin, leading to elevated total
bilirubin. The patient develops jaundice (yellowing of the skin and eyes). Urine may contain
bile pigments and may have bile salts.
3.2.3. Jaundice due to post-hepatic causes
Causes: bile duct obstruction (common bile duct stones, bile duct worms, bile duct
polyps, etc.), pancreatic head tumor, Oddi sphincter spasm, etc.
In this case, Hb metabolism in the spleen and liver is completely normal, but the liver-
intestine cycle is interrupted, preventing bile from flowing into the intestine. Bile duct
pressure increases, conjugated bilirubin accumulates in the liver and bile ducts, spills into the
blood, and increases in the blood.
Result: elevated conjugated bilirubin, jaundice (yellowing of the skin and eyes). Dark
urine due to increased bile pigments and bile salts, pale stools due to absence of urobilinogen
and stercobilinogen pigments.
DISCUSSION CONTENT
1. The process of hemoglobin degradation (the liver-intestine cycle).
2. Hemoglobin degenerative disorders and their clinical significance.
 Chapter 12
NUCLEIC ACID METABOLISM

OBJECTIVES
After completing this chapter, students will be able to:
1. Describe the general degradation of nucleic acids.

2. Describe the degradation of purine nucleotides and pyrimidine nucleotides using

diagrams.
3. Explain the origin of the raw materials for purine nucleotide synthesis.
4. Describe the synthesis of pyrimidine nucleotides.

All cells in living organisms have the ability to synthesize nucleic acids without
requiring raw materials from food. Therefore, the nucleic acid content of food is not
particularly important for animals. By studying the rate of nucleic acid renewal in the body, it
has been concluded that ribonucleic acids (RNA) are characterized by intense exchange and
rapid renewal, with RNA levels increasing rapidly in cells along with intense protein
synthesis, while DNA renewal in tissues is much slower and is observed during cell growth,
regeneration, and division.

1. NUCLEIC ACID DEGRADATION

1.1. General degradation
In animal tissues, nucleic acid hydrolysis occurs.
Nucleic acids are hydrolyzed by nuclease enzymes, which belong to the
phosphodiesterase class. Nuclease acts by hydrolyzing and breaking the phosphodiester bonds
between mononucleotides, releasing these mononucleotides in their free form. There are two
types of nuclease:
ADNase (Deoxyribonuclease)
ARNase (Ribonuclease)
Based on their function, each type of enzyme is divided into two categories:
- Endonuclease: breaks down the phosphodiester bonds within a polynucleotide chain,
creating short nucleotide fragments.
- Exonuclease: hydrolyzes mononucleotides at the terminal ends of the chain, releasing
free mononucleotides. There are two types of exonuclease:
+ Exonuclease 1: Cuts phosphodiester bonds at the 3' end
+ Exonuclease 2: Cuts phosphodiester bonds at the 5' end.
 Figure 12.1. Hydrolysis of nucleic acids by enzymes

Thus, on the polynucleotide chain, through the combined action of these two enzymes,
the molecule is completely degraded into free mononucleotides.
Next:

Thus, the final hydrolysis products of nucleic acids are:

- H3PO4: either reused or excreted in urine as inorganic or organic phosphate salts.

- Pentose sugar: metabolized via the carbohydrate pathway.

- Nitrogen bases: degraded via a separate pathway to produce urea and uric acid, which
are then excreted (primarily via the kidneys and urinary tract).
1.2. The degradation of nitrogenous bases
There are two types of nitrogen bases:
- Purinic nitrogen bases.
- Pyrimidine nitrogen bases.
They degrade via two different pathways.
1.2.1. The degradation of purine nitrogen bases
In humans and higher animals, the degradation of purine nitrogen bases produces the product
uric acid and excreted.
In lower animals, uric acid is further metabolized into allantoin by the enzyme uricase
and excreted. In bony fish, the final product is allantoate. In cartilaginous fish and
amphibians, the final product is urea. In marine invertebrates, the final product is NH3 (Figure
12.2).
In bacteria, degradation produces urea as the final product. Subsequently, urea is further
hydrolyzed to produce CO₂ + NH₃.

Figure 12.2. Schematic diagram of purine nitrogen base degradation

 In addition to degradation from adenine and guanine, the degradation of adenine also
occurs from its nucleoside, adenosine.
Thus, in humans, the degradation product is uric acid. Uric acid is excreted by the
kidneys through filtration. Normally, blood uric acid levels range from 3 to 6.5 mg/dl. Blood
acid levels vary depending on diet, increasing slightly with a high-protein diet, especially
foods containing animal organs, and increasing pathologically in diseases related to nucleic
acid metabolism disorders, particularly in gout, leukemia, or in cases of reduced kidney
filtration function.
1.2.2. Degradation of pyrimidine nitrogen bases
The degradation of pyrimidine nitrogen bases occurs in the liver according to the following
diagram:

Figure 12.3. Diagram of pyrimidine nitrogen base degradation

 Fate of the products: - β-alanine: degraded via the amino acid pathway.
- β-aminoisobutyric acid: degraded via a separate pathway.
2. SYNTHESIS OF NUCLEIC ACIDS
2.1. Synthesis of pentose sugars
- Ribose: formed as ribose-5-phosphate, due to the breakdown of glucose via
the hexose monophosphate pathway.
- Deoxyribose: synthesized via the reaction:

2.2. Synthesis of nitrogen bases

In the body, nitrogen bases are not synthesized separately; their synthesis is linked to
nucleotide synthesis.
2.2.1. Purine nucleotide synthesis
Using radioactive isotopes 14C and 15N, the origins of the elements forming purine bases
were determined as follows:

- Glycine provides C at positions 4 and 5

and N at
position 7;

- Formic acid provides C at positions 2 and

8;

- Glutamine provides N at positions 3 and 9;

- Free CO2 provides C at position 6;

- Aspartic acid provides N at position 1.

Figure 12.4. Origin of purine base-forming elements

* Synthesis process:
Purin nucleotides are synthesized based on a ribose-5-phosphate molecule, consisting of
the following four stages:
- Stage 1: Formation of glycinamide ribosyl-5'-phosphate (glycinamide ribonucleotide).
- Stage 2: Formation of the purine imidazole ring.

- Stage 3: Formation of the pyrimidine ring of purine and the formation of inosine acid.

Stage 4: Conversion of inosine acid into adenine acid (AMP) and guanine acid (GMP).
 Thus, through these four stages, purine nucleotides are formed in the form of nucleotides
monophosphate nucleotides (AMP and GMP). Subsequently, they are converted into ATP and GTP.
The synthesis of purine nucleotides is regulated by a feedback mechanism; high
concentrations of the formed purine nucleotides inhibit the enzyme system involved in their
synthesis.

Figure 12.5. Schematic diagram of purine nucleotide synthesis

2.2.2. Pyrimidine nucleotide synthesis

Unlike purine nucleotide synthesis, pyrimidine nucleotides are synthesized in their complete form.
Before attaching the phosphate group. The raw materials for the synthesis are: aspartic acid, CO₂, NH₃.
The synthesis process consists of two stages.
- Stage 1: Formation of orotic acid
Orotic acid is an important intermediate in the synthesis of pyrimidine nucleotides.

- Stage 2: Formation of pyrimidine nucleotides

 * Synthesis diagram

Figure 12.6. Synthesis diagram of pyrimidine nucleotides

2.2.3. The pathway for "recycling" nitrogen bases

In addition to synthesizing new ribonucleotides as described above, the body also has
another synthesis method, which is to reuse free nitrogen bases originating from nucleic acid
degradation to synthesize new nucleotides.
2.2.4. Deoxyribonucleotide synthesis
Currently, there are several hypotheses explaining this process:
- Deoxyribonucleotides are formed through the reduction of ribonucleotides, catalyzed
by the enzyme ribonucleotide reductase, with the hydrogen donor being NADPH H+and the
involvement of the hydrogen carrier thioredoxin.
 - Or:

2.3. SYNTHESIS OF NUCLEIC ACID

2.3.1. DNA Synthesis (Replication)
[Link]. The synthesis process
DNA is the genetic information basis of all organisms (except RNA viruses). This
information is preserved during cell division, which occurs after DNA synthesis. Therefore,
DNA synthesis requires the accurate replication of the genetic information contained in the
parent cell.
DNA synthesis is a replication process. Based on the structure proposed by Watson and
Crick, each strand of the DNA molecule is used by the cell as a template to synthesize the
second strand according to the complementary base pairing rule, forming a daughter DNA
molecule. One of these two strands is the parent DNA strand.
The raw materials used by the cell to synthesize DNA are deoxyribonucleotide
triphosphates, based on the template of a single parent DNA strand, with the participation of
the polymerase enzyme, DNA polymerase.
[Link] [Link]

[Link] DNA polymerase [Link] + (n1 + n2 + n3 + n4) ℗ ~ ℗

DNA
[Link] DNA primer, [Link]

[Link]
Mg²⁺,K⁺ [Link]
(polymerization)
In reality, this synthesis is very complex and involves many enzymes.
First, the starting point of the replication process is recognized on the DNA molecule.
This recognition point is due to the combination of a recognition protein with a DNA segment
(ori). Next, the double strand is unwound by topoisomerase, then helicase breaks the hydrogen
bonds between the two strands of DNA, followed by SSB proteins (single strand binding
proteins) attaching to the DNA to keep the strand structure from becoming double-stranded
again. After unwinding and breaking the hydrogen bonds, the region with a "Y" structure is
called the DNA replication fork. At this location, two new DNA strands are synthesized in the
5' to 3' direction with
catalyzed by a multienzyme system called polymerase, in which one strand is synthesized
continuously (fast strand), while the other strand is synthesized discontinuously in the
opposite direction of the unwinding enzyme (slow strand) based on the template of the
parental DNA. The synthesis of the slow strand creates Okazaki fragments. At the end of the
synthesis process, these Okazaki fragments are joined together by the DNA ligase enzyme.

Figure 12.7. DNA Synthesis

[Link]. DNA Repair
Research on the replication process in E. coli shows that the DNA error rate after
synthesis is approximately 10-9 – 10-11 . The reason for this very low error rate is that cells
have a repair enzyme system.
There are many causes of DNA damage during synthesis, such as environmental factors,
physical factors, chemical factors, etc. When these factors affect the synthesis process, they
can lead to:
- Changes in one or two nitrogen bases;
- Deletions in the synthesized DNA;
- The formation of abnormal bonds between nitrogen bases within the same strand or
between two strands. The DNA repair enzyme system after replication consists of four
groups:
- The enzyme system that corrects mismatched base pairs during replication.
- Enzyme systems that repair errors on a base-by-base basis (missing, extra, or incorrect bases).
- The enzyme system that repairs by cutting nucleotide segments.
- The repair enzyme system cleaves the double strand vertically.
2.3.2. RNA synthesis (transcription)
RNA synthesis is the process of transferring genetic information from DNA to
synthesized RNA molecules. This process is based on the same principle as DNA replication,
with synthesis occurring in the5'to3'direction and ATP providing energy.
Studies on mRNA synthesis show that mRNA is synthesized in the cell nucleus through the
polymerization of mononucleotide triphosphates, in the presence of DNA, with the enzyme
DNA-dependent RNA polymerase.
[Link] [Link]

[Link] DNA-dependent RNA polymerase [Link]

RNA + (n1 + n2 + n3 + n4) ℗ ~ ℗
[Link] [Link]
Mg²⁺
DNA template,

[Link] (polymerization) [Link]

Figure 12.8. RNA synthesis

Reverse transcription phenomenon:

In some animal viruses (RNA viruses), the RNA, together with the viral RNA reverse
transcriptase enzyme, has the ability to reverse transcribe to create DNA. When a cell is
infected with this virus, the viral reverse transcriptase enzyme catalyzes the synthesis of DNA
from the viral RNA to form an RNA-DNA hybrid. In some viruses (retroviruses), this hybrid
can integrate into the host cell's genome and continue to divide with the host cell's DNA,
altering the normal structure of the host cell's DNA (the mechanism by which some viruses
cause cancer).
Clinically, substances used in the treatment of viral infections (antivirals) are those that
inhibit this reverse transcription process, such as AZT (3-azido-2,3-dideoxythymidine), an
HIV (human immunodeficiency virus) treatment drug that strongly inhibits HIV replication.
DISCUSSION CONTENT

1. General degradation of nucleic acids.

2. Degradation of purine and pyrimidine nitrogen bases.
3. Sources of raw materials for purine nitrogen base synthesis.
4. Origin and raw materials for purine nucleotide synthesis.
5. The synthesis process of purine nucleotides.
 Chapter 13
PROTEIN SYNTHESIS
OBJECTIV
ES
After completing this chapter, students will be able to:
1. Describe the factors involved in protein synthesis and their main roles.
2. Describe the process of protein synthesis.

Protein synthesis, also known as translation, is the process of translating genetic

information from mRNA into the amino acid sequence (primary structure) of the synthesized
protein molecule.
For living organisms, proteins perform numerous vital functions such as enzymatic
roles, structural components, protective functions, etc. Therefore, protein biosynthesis is of
paramount importance to life.
On the other hand, proteins are highly specific and genetically transmitted between
generations. Therefore, the process of protein biosynthesis is very complex and involves many
factors.

1. FACTORS INVOLVED IN PROTEIN SYNTHESIS

1.1. DNA (deoxyribonucleic acid)
DNA is concentrated in the cell nucleus, serving as the material basis of heredity and
determining the specific structure of the protein molecule to be synthesized. Therefore, DNA
determines the biological properties and functions of proteins. Any changes in the genetic
structure of DNA will lead to changes in the structure of the synthesized protein molecule.
Transcrip mRNA
Translat
DNA tion ion Polypeptide chain

1.2. RNA (ribonucleic acid)

There are three types of RNA: mRNA, tRNA, and rRNA.
1.2.1. Messenger RNA (mRNA)
Synthesized in the cell nucleus based on the DNA template. Therefore, mRNA is the
carrier of genetic information from DNA to the protein molecule that will be synthesized.
Regarding structure: there are 3 types of codes on mRNA:
- Start codon (AUG): in bacterial cells, this is where formylated methionine (f Met) is attached
- Extension codon: These are specific polynucleotide sequences that determine the order
of amino acids in the protein molecule to be synthesized, where every three consecutive
nucleotides code for one amino acid.
 - Stop codon (UAA, UAG, UGA): These are nonsense codons that do not encode
any amino acids.
mRNA has a short lifespan, approximately a few days in humans.
1.2.2. Transfer RNA (tRNA)
Also known as soluble RNA because they are dissolved in the cytoplasmic fluid of cells.
Role: specifically transports activated amino acids to the ribosome, where protein
synthesis occurs. Approximately 40 types of tRNA have been identified.

Structure: tRNA consists of a 3'

Amino acid
A
polynucleotide chain comprising 75–85 C
5’ C
mononucleotides twisted into a "hairpin"
shape. In which:
- The 5' end is shorter, typically a
guanine nucleotide (GMP)
- The 3' end contains a free nucleotide
triplet ACC, which is common to all tRNAs,
and is the binding site for the specific amino
acid transporter.
During transport, the amino acid binds to The 3' codon
the -OH group of adenine. Figure 13.1. Structure of transfer RNA
- The anticodon triplet (anticodon): is the triplet of nucleotides corresponding to the
amino acid that the tRNA will specifically transport. When the tRNA binds to the specific amino
acid at the 3' end, it forms an aminoacyl-tRNA (aa-tRNA) complex. This complex is transported to the
ribosome, where the anticodon recognizes the corresponding code on the mRNA according to the
base-pairing rule.
Additionally, there are several sites on tRNA that perform different functions.
1.2.3. Ribosomal RNA (rRNA)
It is synthesized in the nucleus based on a single strand of DNA. ARNr accounts for the
largest amount (about 80%), and it usually combines with a special type of protein to form
ribosomal particles (ribonucleoproteins), which is where protein synthesis occurs.
Ribosomes in all cells have a structure consisting of two subunits. In animals, these are
60S and 40S, which combine to form an 80S ribosome. In bacteria, these are 50S and 30S,
which combine to form a 70S ribosome.
On the larger subunit (50S) in bacteria, there are two functional sites:
- Site A (right): where the activated amino acid delivered by tRNA (aa-tRNA) binds.
- Position P (left): where the peptidyl-tRNA chain is accepted. The
30S subunit has a groove for mRNA binding.
 During biosynthesis, multiple ribosomes bind to a single mRNA molecule to form
polysomes.

Ribosom Amino
e acid

Uracil

Messenger Ribosomal Transfer RNA

RNA RNA (tRNA)
(mRNA) (rRNA)

Figure 13.2. Types of RNA

1.3. Enzymes
- Aminoacyl-tRNA synthetase: catalyzes the reaction forming the aa-tRNA complex, an enzyme
specific to both amino acids and tRNA.
- Peptidyl synthetase and peptidyl transferase: present in the 50S subunit of the
ribosome, catalyzing the reaction forming the peptide bond and transferring the peptidyl
during biosynthesis.
1.4. Energy and ions
1.4.1. Energy: includes ATP, GTP
- ATP: supplies energy for amino acid activation and attaches activated amino acids to tRNA.
- GTP: provides for the binding of aa-tRNA to the A site of the ribosome, forming peptide
bonds and transferring the peptidyl.

1.4.2. Ions
Includes Mg²⁺, K⁺, NH₄⁺.
Role: Stabilize ribosomal states and bind activated amino acids to the ribosome.
1.5. Initiator, elongator, and terminator factors
Nature: All are proteins; they are not permanently present on the ribosome. Includes:
- Initiation factor (IF): required for the formation of the initiation complex.
- Elongation factor EF: required for peptide bond formation and peptide chain
elongation.
- Release factor (RF): required for the termination of polypeptide chain synthesis.
1.6. Materials
20 free amino acids in the cell.

2. PROTEIN SYNTHESIS (IN PROSYSTEMIC CELLS – PROKARYOTES)

Involves two processes:
2.1. Amino acid activation (outside the ribosome)
The catalytic enzyme is aminoacyl-tRNA synthetase, which acts in two stages:

- Amino acid activation: Mg²⁺participates in the formation of the fMet-tRNA complex

Acyl phosphate bonding

- Formation of the aminoacyl-tRNA complex:

The amino acid is attached to the specific tRNA at the amino acid binding site, and the bond
between the tRNA and the amino acid is an ester bond.
The aa-tRNA complex formed will be transferred to the ribosome, where protein synthesis
is taking place.

With methionine as the starting amino acid, it must first be formylated to form f-Met.
Then f-Met is activated to form fMet-tRNA.
Thus, tRNA serves as the carrier for amino acids to the ribosome, where protein synthesis
occurs.
2.2. Polypeptide chain synthesis (occurring on the ribosome)
This is essentially the process of reading the code on mRNA in the5'→3'direction (corresponding
to the start codon AUG, GUG) until the stop codon (UAA, UAG, UGA).
Amino acids recognize their encoded sequences on mRNA through the complementary
codons on tRNA, following the base-pairing rules.
The synthesis process consists of three stages: initiation, elongation, and termination.
2.2.1. Initiation phase
The activation of the initial amino acid leads to the formation of the fMet-tRNA complex by the
enzyme fMet-tRNA synthetase.
In E. coli bacteria, the initial amino acid of the chain, which has been activated as formyl-
methionine (f-Met), is encoded by the start codon on the receiving mRNA.
The process is carried out:

Figure 13.3. Initiation phase

- The 70S ribosome in its inactive state splits into two subunits, 50S and 30S.

- IF (initiation factor) appears.

- An mRNA molecule binds to the 30S subunit of the ribosome at the binding groove.

- fMet-tRNA delivers fMet to bind to the mRNA at the start codon (AUG, GUC).

- The 50S subunit binds to the 30S subunit to form the active 70S complex and completes the
initiation phase.
initiation phase.
 - IF detaches from the ribosome.

The energy supplied for this process is GTP.

Thus: at the end of the initiation phase, fMet-tRNA is located at the P site (50S) of the
ribosome, and the A site is empty, containing a free nucleotide triplet.
2.2.2. Extension phase
Involves cycles of sequentially attaching amino acids from the first to the last according
to a specific sequence encoded on mRNA to form a specific polypeptide chain. Each attachment
cycle consists of three steps with the involvement of EF (elongation factor).
- Step 1: aa1 is activated and binds to the specific tRNA. The aa1-tRNA complex binds to the
A site of the ribosome.
- Step 2: A peptide bond is formed between fMet-aa1 by the peptidyl synthetase enzyme.
A dipeptidyl-tRNA is formed.

- Step 3: Translocation.
The 70S ribosome moves a distance corresponding to one codon on the mRNA via the
peptidyl transferase enzyme. The dipeptidyl-tRNA moves from A → P. At this point, the now-empty
A site continues to accept aa2.
GTP provides the energy for amino acid acceptance, peptide bond formation, and
peptidyl transfer.
The cycle continues until the amino acid-tRNA, the polypeptide chain is completed, and it is
located at the P site of the ribosome.

Figure 13.4. Translation

2.2.3. Termination phase
When position A encounters a stop codon (a nonsense triplet), no aa-tRNA will bind to the
ribosome. EF dissociates from the ribosome.
The termination factor RF binds to the ribosome, promoting the release of the synthesized
polypeptide chain.
fMet detaches from the polypeptide, completing the synthesis of the polypeptide chain
(primary structure).
The mRNA detaches from the 30S subunit. The 70S ribosome dissociates into 50S
and 30S subunits. The synthesis cycle begins again.

Figure 13.5. Elongation and termination phases

* Protein synthesis in eukaryotic cells:

The process of protein synthesis in eukaryotic cells occurs in a manner similar to that in
prokaryotic cells
, differing only in a few details:
- mRNA synthesis:
+ In eukaryotic cells: synthesis occurs in the cell nucleus, and after synthesis, it must
undergo a process of completion.
+ In prokaryotic cells: it occurs in the cytoplasm, so protein synthesis is carried out
immediately after transcription ends.
 - Requires more factors to participate, each stage becomes more complex.
- The ribosome is 80S, consisting of two subunits, 40S and 60S.
- It begins with methionine without formylation. Therefore, the initiation complex is Met-
tRNA.

- The of eukaryotic cells is monocistronic, while that of prokaryotic cells is

mRNA
polycistronic. Therefore, eukaryotic mRNA has only one starting point and serves as a
template for the synthesis of only one protein molecule. In contrast, eukaryotic mRNA has
multiple initiation sites and can serve as a template for the synthesis of multiple proteins
simultaneously, often related to each other within an operon.
3. POST-TRANSLATIONAL MODIFICATION OF THE POLYPEPTIDE CHAIN
After synthesis, the polypeptide chain must undergo modification (processing) to form
the final protein molecule with biological function.
The post-translational modification process includes:
- The fMet type in prokaryotic cells, or Met in eukaryotic cells and some amino acids
with terminal –NH2 groups.
- Removal of the signal peptide segment (15-22 amino acids) specific to the protein to be
synthesized.
- Modify specific amino acids such as phosphorylating the –OH groups of serine,
threonine, and tyrosine (milk casein).
- Removing certain peptide segments to create biologically active proteins (converting
pro-enzymes into active enzymes).
- Add certain functional groups to create functional proteins, such as attaching HEM in
hemoglobin, cytochrome, etc.
- Forming intra-chain or inter-chain cross-links to create stable spatial structures
(secondary structures) such as disulfide bonds, hydrogen bonds, salt bridges, etc.
4. SOME ISSUES IN PROTEIN BIOSYNTHESIS
- Protein distribution: After synthesis, proteins are distributed to different organelles
within the cell; some may be transported outside the cell, such as liver cells releasing albumin
and fibrinogen into the plasma.
- Antibiotics inhibit protein synthesis in bacteria, causing them to stop growing. Each
antibiotic acts on one or more steps of the synthesis process.
 Table 13.1. Effects of certain antibiotics on bacterial protein synthesis
Inhibitor Effect
Actomycin Binds to DNA, inhibiting mRNA synthesis.
Tetracycline Inhibits the binding of aminoacyl-tRNA to the A site of the ribosome.
Inhibits the conversion of the initiation complex into the elongation
Streptomycin complex (step 1),
causing misreading of the code.
Chloramphenicol Inhibits the peptidyl synthetase reaction (peptide bond formation) (step 2).
Erythromycin Inhibits peptidyl transfer (step 3).

5. REGULATION OF PROTEIN SYNTHESIS (related chapter and regulation of

metabolism section 2.2.)

DISCUSSION CONTENT
1. Factors involved in protein synthesis and their primary roles in the synthesis process.
2. Protein synthesis in prokaryotic cells, comparing protein synthesis in prokaryotic and
eukaryotic cells.
3. Post-translational modification of polypeptide chains, mechanisms of action of certain antibiotics
on protein synthesis in bacteria.
Chapter 15 Acid-base balance

Chapter 15
ACID-BASE EQUILIBRIUM
OBJECTIV
ES
After completing this chapter, students will be able to:
1. Describe the buffering systems present in the body, and the buffering mechanisms of the
two main buffering systems: bicarbonate and hemoglobin.
2. Analyze the role of the lungs and kidneys in regulating acid-base balance.
3. Describe conditions of acid-base imbalance.
4. List the key parameters for evaluating acid-base imbalance conditions and their clinical
significance.

1. OVERVIEW
Acid-base balance refers to the stability of pH or hydrogen ion concentration H⁺in the body.
To sustain life, the body's fluid environment (internal environment) must remain in a
relatively constant state, in which electrolyte and acid-base balance play a crucial role.
Maintaining the stability of the internal environment is essentially a "dynamic" process.
In reality, acid-base balance in the internal environment is constantly threatened by exogenous
factors (substances entering the body through food, drink, or injections) or endogenous factors
(substances produced during metabolism), but it is quickly restored. This restoration is
achieved through the activity of the body's buffering systems and excretory organs.
Acid-base imbalance disorders are common clinical conditions that can directly affect
many metabolic processes, including the patient's life. Therefore, detecting and restoring acid-
base balance is one of the critical issues in diagnosis and treatment.

2. BASIC PRINCIPLES OF ACID-BASE EQUILIBRIUM

2.1. The Concept of pH
The acid or base state of a solution is determined by the concentration of H+and OH-ions.
One of the properties of water, an essential component of living organisms, is its ability
to dissociate.

According to the law of mass action, at equilibrium we have:

 KH₂O = 1.8 × 10⁻⁶ (16)
The dissociation constant of water is very small. Therefore, it can be considered that this
dissociation does not significantly change the number of water molecules in a unit volume.
The concentration of pure water is 1000/18 = 55.5 mol/liter.
From equation (2), we have:
[H+] [OH-] = KH2O . [H2O]
[H+] [OH-] = 1.8 ×10⁻⁶× 55.5 [H+]
[OH-] =10⁻¹⁴

In pure water, the concentration of H+ions equals the concentration of

OH-ions, or: [H+] = [OH-] =10⁻⁷mol/L

Solutions with an H+concentration greater than10⁻⁷mol/L are called acidic

solutions. Solutions with an H+concentration less than10⁻⁷mol/L are called basic

solutions.
For simplicity, the concentration of H⁺is determined by its negative logarithm value and
is called pH.

pH = -log [H⁺]

According to Bronsted's theory:

Substances capable of donating H⁺ions are acids.
Example:

Conversely, substances capable of accepting H+ions are bases.

Example:

Thus, when an acid is present in a solution, the H+concentration of that solution

will increase. Conversely, when a base is present, the H⁺concentration will decrease.
Some substances may exhibit acidic or basic properties depending on the pH conditions
of their environment.
 Example: (basic environment)
(acidic environment)
Substances with such properties are called amphiprotic. In the body, many substances
are amphiprotic and play important biological roles. Examples include proteins, phosphoric
acid esters, amino acids, and certain other organic acids.
2.2. Buffer solutions and buffer systems
2.2.1. Definition
A buffer solution is a solution that resists changes in pH when a strong acid or a strong
base is added to it.
Composition of a buffer solution: a mixture containing a weak acid and a base derived
from that weak acid or its anion (conjugate base). They form a buffer pair (or buffer system).
Example: a mixture of acetic acid and sodium acetate.
In the body, biological buffer systems play a crucial role in preventing pH fluctuations.
Plasma is a highly effective buffer solution that maintains a constant blood pH (approximately
7.38–7.42).
2.2.2. Buffering mechanism
- When a strong acid is added to a buffer solution: the base portion of the buffer system
(the anion of the weak acid) will combine with the strong acid to form a weakly dissociated
acid. Thus, very few H⁺ ions are produced, so the pH is not significantly affected.

- Conversely, when a strong base is added: this base combines with the weak acid
component of the buffer system, the base is neutralized, forming an anion of the weak acid.
Therefore, it also does not significantly change the pH.

3. THE BUFFER SYSTEMS IN THE BODY AND THEIR BUFFERING ROLES

In the body, different fluids have different pH levels, for example:
- pH of arterial blood: 7.38 - 7.42.
- pH of venous blood: slightly more acidic.
- Extracellular fluid: 7.40
- Intracellular fluid: 6.7.
The pH of these fluids is always relatively constant. This constancy is due to the role of
buffer systems and excretory organs (lungs, kidneys).
3.1. The body's buffering systems
3.1.1. Buffer systems in the blood
-
- Bicarbonate buffer system: H2CO3/HCO3 .
This buffer system is primarily found in plasma, is the most important buffer system in
the blood and the entire body, accounting for 53% of buffer capacity. This buffer system has
maximum buffering capacity when the ratio H2CO3/HCO3-= 1/20.
- Phosphate buffer system:
Consists of two systems: KH₂PO₄/K₂HPO₄ (mainly found in red blood cells)
and NaH₂PO₄/Na₂HPO₄ (mainly found in plasma).
However, in plasma, the role of the phosphate buffer system is not significant because
the concentration of HPO42-ions in the blood is very low (approximately 2 mEq/liter).
- Hemoglobin buffer system (red blood cells). Consisting of: HHb/KHb and HHbO2/KHbO2.
- Protein buffer system: protein/proteinate.
3.1.2. Buffer systems in tissues
Tissues primarily have bicarbonate and phosphate buffering systems, along with a protein
buffering system; there is no hemoglobin buffering system.
3.1.3. Intracellular buffering systems
Intracellular fluid contains buffering systems similar to those in plasma and interstitial
fluid. Since the protein and phosphate concentrations in intracellular fluid are higher than in
interstitial fluid, the protein and phosphate buffering systems play a significant role in this
region.
On the other hand, there is a dynamic exchange of H+ions, some electrolytes, and anions
between the intracellular and extracellular regions. Therefore, these regions provide
significant mutual support in the buffering process of the entire body.
3.2. Buffering mechanism
Take the bicarbonate and Hb buffer systems as examples.
3.2.1. Buffering mechanism of the bicarbonate buffer system: H2CO3/ HCO3-
- When a strong acid enters:
H⁺(from the acid) + HCO₃⁻ H2CO3 H2O + CO2
Thus, to counteract the acid, a certain amount of CO2 is produced, which is then exhaled
through respiration.
- Conversely, when a strong base enters:
H2CO3 H++ HCO3-
H⁺+ OH⁻(from the invading base) → H₂O
 To neutralize the base, a certain amount of HCO3-is produced, and this excess HCO3-is
excreted by the kidneys.
Thanks to this buffering mechanism, the body's pH remains relatively stable even when
a strong acid or a strong base enters the system.
3.2.2. The mechanism of the Hb buffer system
The Hb buffer system is the most important buffer system found in red blood cells. The
buffering mechanism of the Hb buffer system is closely related to gas exchange in the lungs
and tissues.
- In the lungs:

CO₂ produced is excreted via the respiratory tract, while HbO₂ is transported via arterial
blood to tissues.

- In tissues:
HbO₂ Hb + O₂

CO2 (produced by tissue metabolism) + H 2 O H 2 C O 3 H++ HCO3-Hb + H+

H Hb
HHb and HCO3- travel through the venous blood to the lungs, and the process continues.

Thus, the buffer role of hemoglobin is to participate in the process of eliminating CO2
produced by metabolic processes in respiratory tissues. This mechanism is illustrated as
follows:

Lungs Blood Tissue

Exhalation (formed by metabolism)

Figure 15.1. Mechanism of action of the Hb buffer system
4. THE ROLE OF THE LUNGS IN REGULATING ACID-BASE BALANCE
The lungs regulate acid-base balance by regulating the partial pressure of carbon dioxide
(PCO₂) in the blood and extracellular fluids.
Under normal conditions, we have:
- In plasma:

The CO2 dissolvedin plasma is the amount of CO2 produced by tissue metabolism, which is
transported into the blood and extracellular fluids.
- In the lungs:
Alveolar C O ₂ Dissolved CO₂ H₂CO₃ H⁺+ HCO₃⁻

According to Henry's law: the concentration of a dissolved gas in a solution is directly

proportional to the partial pressure of that gas exerted on the surface of the solution. Or:

CO2 dissolved = α. PCO2 in the alveoli.

Alveolar PCO₂: the partial pressure of CO₂ in alveolar air. α: the solubility
coefficient of CO₂ in plasma, at 37°C α = 0.03 mmol/L. On the other hand,
due to the circulatory exchange process:
Alveolar PCO₂ = Arterial PCO₂.

From equation (1), when the equation reaches equilibrium, we have:

dissolved

(alveoli) (arterial)

(arterial)

(artery)

Or at 37°C
(artery)
 This is the basic equation for expressing the dependence of pH, or acid-base balance
- of a living organism.
Thus, the pH of blood and extracellular fluids is directly proportional to the concentration of
bicarbonate
(HCO₃⁻) 3and inversely proportional to the partial pressure of carbon
2 dioxide
2 (PCO₂) in arterial blood.

This is also an expression used for extrapolation, through which to evaluate acid-base
balance conditions in the body.
* The role of the lungs in regulating acid-base balance
The role of the lungs in regulating acid-base balance is to maintain a constant partial
pressure of carbon dioxide (PCO₂) in arterial blood. Specifically:
- The lungs' CO2 elimination function under normal conditions

Figure 15.2. Lung function under normal conditions

The human body functions as an open system. Under normal conditions, the amount of
CO2 produced by tissue metabolism is dissolved into the blood plasma as dissolved CO2, and an
equal amount is exhaled through respiration. Thus, the amount of CO2 produced equals the amount
exhaled, leading to stable blood PCO2 and stable pH.
- In a state of acidosis
When strong acids enter the body, a certain amount of CO2 is produced due to acid
neutralization, causing an increase in blood PCO2. To counteract this increase in CO2, the lungs
must increase CO2 excretion through rapid, deep breathing (CO2 excretion > CO2 production) so
that arterial blood PCO2 increases but remains stable and pH does not change.
 Figure 15.3. Lung activity in an acidic state

- In a state of alkalosis
In a state of base overload: the concentration of H+ions decreases due to base
neutralization, leading to a decrease in H2CO3 concentration, which in turn reduces the
concentration of dissolved CO2 in the blood and lowers plasma PCO2. Thus, to counteract the
decrease in plasma PCO₂, the lungs reduce CO₂ excretion by breathing slowly and shallowly
(CO₂ excretion < CO₂ production), causing blood PCO₂ to decrease but remain stable and pH
to remain stable.

Figure 15.4. Lung function in a basic state

 Thus: The lungs regulate acid-base balance by maintaining stable blood PCO₂ and
extracellular fluids through adjusting breathing rate: either rapid, deep breathing (i.e.,
increased CO₂ excretion) in acidotic states, or slow, shallow breathing (i.e., reduced CO₂
excretion) in alkalotic states.

5. THE ROLE OF THE KIDNEYS IN ACID-BASE BALANCE REGULATION

It can be said that the kidneys are the most important organs involved in regulating the
body's acid-base balance. However, the regulatory role of the kidneys is slower than that of
the lungs, and the acid-base balance regulation of both organs is slower than that of the
buffering systems.
The kidneys regulate acid-base balance through three mechanisms:
- Reabsorb all bicarbonate ions (HCO3-) filtered by the glomerulus back into the blood
plasma.
- Synthesizing a certain amount of bicarbonate daily to compensate for the loss of this
anion due to acid neutralization produced during metabolism, or the amount of HCO3-that may
be lost through extra-renal pathways combined with ammonium salt (NH4+) excretion.
- Excretion of fixed, non-volatile acids and salts produced during metabolism.
5.1. Mechanism of bicarbonate reabsorption (renal tubules)
Under normal physiological conditions, urine does not contain HCO3-ions. All
bicarbonate ions filtered by the glomerulus are reabsorbed back into the plasma by the renal
tubules. Therefore, if the renal tubules are damaged, this reabsorption function will be
affected, potentially leading to a deficiency of HCO3-and causing the body to become acidic.
The ability of renal tubular cells
3 to reabsorb HCO -depends on several factors:

- affects the ability of renal tubular cells to reabsorb HCO₃⁻. This is the kidney's
PCO₂

compensatory mechanism in cases of respiratory acidosis or respiratory alkalosis.

- The reabsorption of HCO3-is also closely related to the reabsorption of Na+in the tubules.
.
- Decreased blood Cl-and K+concentrations increase HCO3-absorption.
As shown in the diagram (Figure 15.5), HCO -is reabsorbed in the renal tubule via two
3
pathways
pathways:
- Dependent on the enzyme anhydrase (30%).
- Not dependent on anhydrous carbocylase (70%), this is an energy-dependent active
transport pathway (the activity of the enzyme HCO3--ATPase).
 Tubular lumen Tubular cells Plasma

Figure 15.5. Reabsorption of HCO -in the renal tubule

3

Active transport
Passive diffusion AC:
Anhydric carbocylase

Thus, under normal conditions, the amount of HCO3-filtered from the plasma into the renal
tubule lumen as NaHCO3 is equal to the amount of HCO3-reabsorbed by the renal tubule cells back
into the plasma also as NaHCO3.
In a state of metabolic alkalosis, an excess amount of HCO3-is produced, leading to an
increase in blood HCO3-concentration. To counteract this increase, the kidneys cease the
production of HCO3-and may excrete some of the HCO3-filtered by the glomeruli into the urine if
the blood HCO3-level remains high, causing the urine to become alkaline.
5.2. Mechanism of HCO3- regeneration and ammonium salt (NH4+ ) excretion (renal tubules)
Under normal conditions, a certain amount of acid is produced during metabolism
(approximately 50-100 mEq H+ /24 hours), such as sulfuric acid, phosphoric acid, uric acid,
etc., which is released into the blood and extracellular fluids. In the extracellular space, they
are neutralized by HCO3(-),which is the cause of the continuous loss of HCO3(-)in the internal
environment. This lost amount of HCO3(-)is compensated by the HCO3(-)regeneration
mechanism of the renal tubule cells combined with the excretion of NH4(+)salts.
Thus: under normal conditions in the renal tubule cells, HCO3-is constantly regenerated
and released into the blood, accompanied by the excretion of NH4+into the urine, independent of
NH3 concentration.

The excretion of ammonium salts depends on two factors:

 - The concentration of NH3 in the renal tubular cells.
- The pH of the urine.

Tubular lumen Tubular cells Plasma

Figure 15.6. HCO3-formation and NH4+excretion in the renal tubule

5.3. Excretion of non-volatile acids into urine

In the renal tubule, besides NH3, which is an H+acceptor,there are also phosphate salts
(HPO42-), creatinine, and urate. These substances have the ability to accept H+to form their
acidic forms.
Additionally, metabolic products such as organic acids (known as non-volatile fixed
acids) like lactic acid, pyruvic acid, and ketone bodies are directly excreted into the urine.

Thus: the kidneys regulate acid-base balance by maintaining stable concentrations of

-
HCO3 in the blood and extracellular fluids. In cases of acidosis, the renal tubules increase

HCO3(-)production,i.e., increase the excretion of ammonium salts (NH4(+) ). Conversely, in

cases of alkalosis, the kidneys stop producing HCO3(-)and may excrete some of this ion into
the urine if its concentration in the blood is too high.

6. ACID-BASE IMBALANCE
There are many causes of acid-base imbalance.
Categorized into two types of causes:

- Disorders caused by respiratory factors (due to the lungs).

- Disorders due to metabolic causes.
 When the body can self-regulate the disorder, it is called a compensated disorder. When
the body is unable to self-regulate the disorder, it is called a decompensated disorder, which
requires timely detection and treatment.

6.1. Disorders caused by respiratory factors

There are two conditions: - Respiratory acidosis.
- Respiratory alkalosis.

6.1.1. Respiratory acidosis

Includes all causes leading to increased PCO2 in the blood and extracellular fluids.

- Causes that obstruct respiration and reduce ventilation in the lungs, such as asthma
, lung collapse, pleural effusion, pneumothorax, chronic pulmonary heart disease, etc.

- Additionally seen in: premedication, paralysis, high-dose sedation...

Because breathing is obstructed, ventilation is reduced, leading to increased blood PCO2 and
decreased pH
decrease.

6.1.2. Respiratory alkalosis

Includes causes leading to decreased blood PO2 and extracellular fluids. Found in all
conditions of excessive ventilation.
The most common cause is damage to the central nervous system (particularly the
cerebral cortex), which stimulates the respiratory center, such as cerebral hemorrhage, brain
tumor, encephalitis, meningitis, etc. In these conditions, patients breathe rapidly and deeply,
leading to increased CO2 excretion, causing a decrease in blood CO2 levels, reduced PCO2, and
increased pH.

6.2. Disorders caused by metabolic factors

There are also two conditions: metabolic acidosis and metabolic alkalosis.

6.2.1. Metabolic acidosis

Metabolic acidosis is the most common condition, including all diseases that lead to an
excess of non-volatile fixed acids or a deficiency of HCO3-in the blood and extracellular fluids,
resulting in a decrease in blood pH.
Causes:

- Due to reduced excretion: renal failure, tubular disease, causing the kidneys to reduce
excretion of fixed acids produced by metabolism, such as uric acid, lactic acid, etc.

- Due to metabolic disorders or increased metabolism: diabetes, cancer, prolonged high fever, etc.
- Due to loss of HCO3-: commonly lost through the digestive tract, such as diarrhea, or duodenal
drainage.
6.2.2. Metabolic alkalosis
These are pathological conditions leading to a deficiency of non-volatile fixed acids,
or an excess of HCO3-in the blood and extracellular fluids.
Causes:

- Excessive vomiting, gastric drainage causing loss of gastric fluid (containing HCl).
- Diseases causing increased aldosterone production.
- Pyelonephritis.
Additionally, mixed disorders may occur.

7. PARAMETERS FOR ASSESSING BALANCE DISORDERS

ACID-BASE
There are many parameters used to assess acid-base imbalance. Below
are some of the main parameters.

7.1. Blood pH
Blood pH is measured using a pH meter on arterial blood samples or arterialized
capillary blood, under conditions where the blood is not exposed to oxygen.
This parameter is of limited significance, so to assess the body's acid-base balance, it
must be combined with other parameters.
Normal arterial blood pH is 7.38–7.42.
Due to the buffering mechanism, many cases have experienced acid-base imbalance to
a certain degree, but blood pH has not yet changed significantly.
7.2. Arterial blood PCO2
This parameter depends solely on lung function, specifically on alveolar ventilation;
arterial PCO₂ is always inversely proportional to alveolar ventilation.
The normal value for arterial PCO₂ is approximately 40 mmHg.

The PCO₂ parameter is used to assess respiratory causes of imbalance.

7.3. Actual bicarbonate (AB: Actual bicarbonate)

This is the actual bicarbonate concentration in the sample, taken in a state where the
blood has not been exposed to air, corresponding to the actual pH and PCO2 of the sample being
measured.
The normal AB value is 25 mEq/L.
This parameter is highly dependent on PCO₂. When PCO₂ increases, it leads to an increase
in AB.
AB assesses disorders caused by metabolic factors.
7.4. Standard bicarbonate (SB)
This is the bicarbonate concentration of blood adjusted to standard conditions: PCO₂ =
40 mmHg, normal PO₂, temperature of 37°C, and hemoglobin fully saturated with oxygen.
The normal value is 25 mEq/L.
SB only changes in cases of disorders caused by metabolism.

7.5. Buffer base (BB)

BB is the total concentration of buffer anions in whole blood (HCO3-, Hb-, protein-, HPO -
…). 4

BB is not significantly influenced by PCO₂, but is affected by the concentration

hemoglobin concentration in the blood.
The normal value of BB = 46 mEq/L.
BB changes in metabolic disorders.

7.6. Base excess, base deficiency (EB: Excess base)

Base excess (or base deficit) is the difference between the patient's base concentration
and that of a healthy individual. In other words, EB is the amount of abnormal excess acid or
base in the blood.
The normal value of EB in a blood sample with pH = 7.4 and PCO2 = 40 mmHg is
0 (more precisely ± 1).
A negative BB value indicates a base deficit or acid excess, while a positive BB value
indicates a base excess or acid deficiency in the blood.
The EB parameter is very important, as it directly indicates the amount of acid or base
that needs to be adjusted for the patient to restore their acid-base balance to normal.
7.7. Total CO2
This value represents the total amount of CO2 in the blood taken under conditions without
exposure to air, with dissolved CO2, H2CO3, and HCO2 measured directly using the Vanslyke
manometer method.
In blood, the concentration of HCO3-is typically 20 times higher than that of dissolved CO2,
so changes in dissolved CO2 are primarily due to changes in HCO3-concentration.
Currently, modern blood gas analyzers in hospitals allow for the determination of all the
above parameters.
Some technical requirements for blood collection for blood gas testing:
- Arterial blood is drawn using a specialized syringe.
 - The machine is calibrated before measurement and measures immediately after blood
collection.

- Avoid: Allowing air to enter the sample; Leaving blood out for too long and not storing it
properly
requirements.
The Davenport diagram can be used to identify acid-base imbalance conditions.

Acidosis Alkalosis

Figure 15.7. Davenport diagram

The Davenport diagram has two axes:

- The horizontal axis is pH (6.9 - 7.7)

- The vertical axis is HCO3 concentration (mmol/l).
The line parallel to the vertical axis represents Hb concentration
(g/dl)
The diagonal line connects the normal Hb concentration to the center of the circle
(normal individual).
The curves represent PaCO2 (partial pressure of CO2 in arterial blood).
 On the graph, there is a circle defined by the parameters in a normal person
: pH = 7.38 - 7.42; PaCO2 = 40 mmHg; HCO3 = 25 mmol/l and Hb = 15 g/dl.
From the two parallel vertical axes, at the points pH = 7.38 and 7.42, the PaCO2 curves
intersect at 40 mmHg and the Hb = 150 g/dl line, forming six regions of acid-base imbalance.
- Region A: Respiratory acidosis (increased PaCO₂).
- Region B: Metabolic alkalosis (increased HCO3-).
- Zone C: Respiratory alkalosis (decreased PaCO2).
- Region D: Metabolic acidosis (decreased HCO3-).
- Region E: Mixed acidosis (increased PaCO₂ and decreasedHCO₃⁻).
- Region F: Mixed alkalosis (decreased PaCO₂ and increasedHCO₃⁻).

DISCUSSION CONTENT
1. Buffer systems in the body, the buffering mechanisms of the two main buffer systems are bicarbonate
and Hb.
2. The role of the lungs and kidneys in acid-base balance regulation.
3. Acid-base imbalance disorders.
4. Factors for assessing acid-base imbalance.
Chapter 16 Water and inorganic substance
exchange

Chapter 16
WATER AND INORGANIC SUBSTANCE EXCHANGE
OBJECTIV
ES
After completing this chapter, students will be able to:
1. Describe the role and distribution of salt and water in the body.

2. Explain the main factors that determine the transport and distribution of water.
3. Describe water transport between regions: plasma and interstitial fluid, inside and
outside cells.
4. Analyze disorders of salt-water exchange.

In the body, water and inorganic substances (called salts) do not play an energy-
providing role like organic substances, but they play an important role in cell and tissue
structure, maintaining cell and tissue activities. Without water and inorganic substances
(salts), life can cease even sooner than without organic food.
Water and salt exchange are always closely related to each other, and they are also
related to the metabolism of organic substances. Salt-water imbalance disorders are common
clinical conditions, diverse and potentially life-threatening, therefore requiring proper
assessment and timely treatment.

1. THE ROLE AND NEED FOR SALT AND WATER IN THE BODY
1.1. The role of salt and water
1.1.1. The role of water
- Water is a component of the body.
Water constitutes the largest proportion of the body's composition. In adults, water
accounts for 55–65% of body weight, with this ratio varying depending on age, gender, and
body type.
+ By age
Table 16.1. Water content by age (percentage by weight)
Age Water content (%)

2-month-old fetus 97

3-month fetus 94

4-month fetus 92

5 months pregnant 85–87

Newborn 66–75

Adults 55–65