SBK3013 :

PRINCIPLES OF BIOCHEMISTRY

Carnitine Deficiencies

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LEARNING DICUSSION
Define Carnitne and role of carnitine in human body
Defintion of Carnitine Deficiency
Dicussion on cause and effects of Carnitine Deficiency
Dicuss on Primary Carnitne deficiency and secondary
deficiency
What is effects of low carnitine
How people with the disorder metabolize muscle glycogen
aerobically?
Dicussion on the significance presence of lipid vacuole in
muscle biopsy
Explain Mitochondrial (beta) -Oxidation Reactions
Synthesis of Triglycerides

Case
Carnitine deficiency
A teenage boy was brought to a hospital as he complaints
that he always get too tired when asked to participate in
the any school activities. The doctor found muscle
weakness in the boys arms and legs. From the muscle
biopsy, the lab pathologist found greatly elevated
amount of triglycerides esterified with primary long
chain fatty acid. They also found significant presence
of lipid vacuoles in the muscle biopsy. What cause
these symptoms?

What is Carnitine?
Carnitine - substance found in
almost every cell in the body, it is
biosynthesized from - amino acids
lysine and methionine.
There are three different forms of
carnitine:
a)L-carnitine
b)acetyl-L-carnitine
c)propionyl-L-carnitine

Role of
Carnitine?
Carnitine -helps the body turn fat into
energy. Our body makes it in the liver
and kidneys and stores it in the skeletal
muscles, heart, brain, and sperm.
The compound plays role in energy
production, as it is responsible for
transporting fatty acids to the
mitochondria. arnitine transports longchain fatty acids into mitochondria
where they are burned (oxidized) to
produce energy.
Carnitine-transports waste and toxic
compounds out of the mitochondria,
preventing their buildup.

Carnitine Deficiency
Carnitine deficiency is one of a
group of metabolic muscle
diseases that interferes with the
processing of food (in this case,
fats) for energy production.
Inborn error of fatty acid transport
caused by a defect in the
transporter responsible for moving
carnitine across the plasma
membrane
When carnitine cannot be
transported into tissues, fatty acid
oxidation is impaired, leading to a
variety of symptoms

What causes carnitine deficiency?

Response to a genetic mutation (gene defect) in the protein
responsible for bringing carnitine into the cell
(primary carnitine deficiency)
May occur secondary to other metabolic diseases
(secondary carnitine deficiency)

Primary Carnitine Deficiency

A person with primary carnitine deficiency
has very low levels of carnitine in the blood
due to a faulty carnitine transporter which
prevents carnitine from getting into the cells
where it is needed.
The primary form of the disorder can be
classified as either "systemic carnitine
decificiency", which affects many organ
systems including the heart and the brain, or
"muscle carnitine deficiency", which is
restricted to vouluntary muscles.

Secondary Carnitine Deficiency

The secondary form of carntine deficiency can
arise secondary to metalobic disorders in the
mitochondria.
Blockage of metabolic pathways in the
mitochondria leads to a build-up of acyl
compounds.
These compounds then bind to carnitine and the
bound complex is then excreted by the kidney,
causing carnitine levels to drop.
Some of these mitochondrial disorders include
cytochrome c oxidase deficiency, mitochondrial
ATPase deficiency, and fatty acyl-CoA
dehydrogenase deficiencies.
In both primary and secondary carnintine
deficiencies, increased dietary intake and
supplements of carnitine can be beneficial.
Although the exact mechanism is unknown, it is
thought that flooding the body with high
concentrations of carnitine assures that some
carnitine are able to get into the cells.

Review primary and secondary causes of carnitine deficiency.

Carnitine is actively transported via OCTN2 into
the cytosol to participate in the shuttling of
activated long chain fatty acids into the
mitochondria where -oxidation takes place.
Carnitine also regulates the Coenzyme A
(CoA)/acylCoA ratio within the mitochondria,
modulation of which reduces accumulation of toxic
acyl-CoA compounds and maintains energy
production.
Free L-carnitine, absorbed from dietary intake or
synthesized in liver and kidney, reaches the blood
stream and the extracellular fluid. Its transport
within cells of various tissues is limited by their
respective uptake capacities. Plasma concentration
of free carnitine is in dynamic balance with
acylcarnitines with the acyl to fee carnitine ration of
0.4 being considered normal . Acetylcarnitine
esters are formed intracellularly during regular
metabolic activity. Long chain acetylcarnitine esters
transport fatty acyl moieties into the mitochondria
(Figure 1). Short and medium-chain acetyl esters,
formed in the mitochondria and peroxisomes,
participate in the removal of organic acids. AcetylL-carnitine is the principal acylcarnitine ester.
Acetyl-L-carnitine participates in both anabolic and
catabolic pathways in cellular metabolism.

Simple explaination of carnitine transporter.

- The main function of carnitine is in the uptake of fatty acids into the mitochondrial matrix for
beta-oxidation. Fatty acyl CoA cannot cross the mitochondrial membrane. At the outer face of
the outer mitochondrial membrane the fatty acyl group is transferred onto carnitine, catalysed by
carnitine acyltransferase 1 (CAT 1). Acyl carnitine is transported across the membranes in
exchange for free carnitine and at the inner face of the inner mitochondrial membrane the acyl
group is transferred onto CoA, catalysed by carnitine acyltransferase 2.

What are the symptoms of carnitine

deficiency?
If confined to muscles, this disease causes:
a)Weakness in the hips, shoulders, and upper arms and legs.
b) The neck and jaw muscles also may be weak.
c)Heart muscle weakness may occur.
In more severe cases, where other tissues are affected, symptoms can include :
a)low blood sugar
b) fatigue
c)vomiting
d)abdominal pain
e)growth retardation
f)low weight
g)enlarged liver
h) brain function abnormalities.

What causes metabolic diseases of muscle?

The metabolic diseases of muscle are caused by genetic defects that interfere with chemical
reactions involved in drawing energy from food.
The fuel molecules derived from food must be further broken down inside each cell before they can
be used by the cells mitochondria (or "engines") to make energy.
Metabolic muscle diseases are caused by problems in the way certain fuel molecules are processed
before they enter the mitochondria, or by the inability to get fuel molecules into mitochondria.
When one of the enzymes in the line is defective, the process goes more slowly or shuts down
entirely.
Our bodies use carbohydrates (starches and sugars), fats and protein for fuel.
Defects in the cells carbohydrate- and fat-processing pathways usually lead to weakness in the
voluntary muscles, but also may affect the heart, kidneys or liver
.

In normal metabolism, food

provides fuel that's processed
inside the cells, producing
energy (ATP) for muscle
contraction and other cellular
functions.
In metabolic myopathies,
missing enzymes prevent
mitochondria from properly
processing fuel, and no
energy is produced for
muscle function.

What are inheritance patterns in metabolic

diseases of muscle?
Most of the metabolic diseases of
muscle are inherited in an autosomal
recessive pattern
It means that a person needs two
defective genes in order to have the
disease.
One copy is inherited from each
parent, neither of whom would
normally have symptoms.
Thus, the disease appears to have
occurred out of the blue, but in
reality, both parents may be carriers,
silently harboring the genetic mutation
(a flaw in the gene).
Many parents have no idea theyre
carriers of a disease until they have a
child who has the disease.

How people with the disorder metabolize

muscle glycogen aerobically?
Energy required for contraction comes
from three main sources:
Creatine phosphate
Glycolysis (anaerobic metabolism)
(cytosol) 6C -------->3C + ATP
Citric Acid Cycle (Aerobic metabolism)
(Mitochondria)
Creatine phosphate serves as a rapid
source of energy, easily donating
inorganic phosphate. It also provides a
limited supply of ATP.

What is the status of research on carnitine

deficiency?
Better diagnosis to allow for earlier identification of at-risk individuals
and earlier treatment
Continued examination of the role of exercise and diet in metabolic
diseases;
Development of animal models of metabolic diseases, both to improve
understanding of the diseases and to test possible treatments;
Development of enzyme replacement therapies
Development of gene therapies.

 During moderate exertion of the body, carbohydrate undergoes aerobic

metabolism.
Under these conditions, oxygen is used and the carbohydrate goes through both the Embden-Meyerhoff
pathway of anaerobic metabolism, in which:
a) glucose is converted to lactate, but before the conversion of pyruvate to lactate
b) pyruvate enters the Krebs Cycle in mitochondria
c)where oxidative phosphorylation results in a maximum extraction of energy from each molecule of
glucose.
d)If there is plenty of oxygen available and the exercise is of low to moderate intensity, then the pyruvate
from glucose is converted to carbon dioxide and water in the mitochondria.
Approximately 42 ATP equivalents can be produced from a single glucose molecule compared to only 4
ATP with
anaerobic metabolism.
A muscle cell has some ATP that it can use immediately, but not very much
only enough to last for about three seconds .
To replenish ATP levels quickly, muscle cells convert a high-energy phosphate compound called creatine
phosphate.
The phosphate group is removed from creatine phosphate by an enzyme
called creatine kinase, and is added to ADP to form ATP.
Together, the ATP levels and creatine phosphate levels are called the phosphagen system. As it works, the
cell turns
ATP into ADP, while the phosphagen rapidly turns the ADP back into ATP. As the muscle continues to
work, the creatine phosphate levels begin to decrease.
The phosphagen system can supply the energy needs of working muscle at a high rate, but only for 8 to
10 seconds.

The significance presence of

lipid vacuole in muscle biopsy
The diagnosis can be established
by demonstrating absence of acid
maltase and phoshphorylase
respectively.
Excess glycogen accumulation in
vacuoles and in the fibers
can be demonstrated by PAS stain
in glycogenoses.
Similarly, lipid accumulation is
Demonstrated in vacuoles or in the
fibers in carnitine deficiency or in
disorders of mitochondrial beta
oxidation.

Basic Concept of Case

Fatty acid are activated on the outer mitochondrian membrane, whereas they are
oxidized in the mitochodrial matrix
A special transport mechanism is needed to carry the long chain acyl CoA
molecules across the inner mitochondrial membrane.
Activited long chain fatty acid are transported across the membrane by
conjugating them to carnitine, zwitterionic alcohol.
The acyl group is transfer from the sulfur atom of CoA to the hydroxyl group
of carnitine to form acyl carnitine.
This reaction is catalyzed by carnitine acyl transferase I ( also called carnitine
palmitoly transferase I) which is bound to the outer mitochodrial membrane .
Acyl carnitine is then shuttled across the inner mitochodrial membrane.
The acyl group is transferred back to CoA on the matrix side of the membrane.
The reaction, which is catayzed by carnitine acyl is transferase II (Carnitine
palmitoly transferase II) is simply the reverse of the reaction that takes place in
the cytosol
Finally, the translocase returns carnitine to the cytosodic side in exchange for an
incoming actyl carnitine

Mitochondrial (beta) -Oxidation Reactions

The process of mitochondrial fatty acid oxidation is termed

-oxidation since it occurs through the sequential removal of
2-carbon units by oxidation at the -carbon position of the
fatty acyl-CoA molecule.
The oxidation of fatty acids and lipids in the peroxisomes
(see below) also occurs via a process of -oxidation.
Each round of -oxidation involves four steps that, in order,
are oxidation, hydration, oxidation, and cleavage.
The first oxidation step in mitochondrial -oxidation
involves
a
family
of
FAD-dependent
acyl-CoA
dehydrogenases. Each of these dehydrogenases has a
range of substrate specificity determined by the length of
the fatty acid. Short-chain acyl-CoA dehydrogenase (SCAD,
also called butyryl-CoA dehydrogenase) prefers fats of 46
carbons in length; medium-chain acyl-CoA dehydrogenase
(MCAD) prefers fats of 416 carbons in length with maximal
activity
for
C10
acyl-CoAs;
long-chain
acyl-CoA
dehydrogenase (LCAD) prefers fats of 616 carbons in length
with maximal activity for C12 acyl-CoAs.

The next three steps in mitochondrial -oxidation involve a

hydration step, another oxidation step, and finally a
hydrolytic reaction that requires CoA and releases acetylCoA and an acy-CoA two carbon atoms shorter than the
initial substrate.
The water addition is catalyzed by an enoyl-CoA hydratase
activity, the second oxidation step is catalyzed by an NADdependent long-chain hydroxacyl-CoA dehydrogenase activity (3hydroxyacyl-CoA dehydrogenase activity), and finally the
cleavage into an acyl-CoA and an acetyl-CoA is catalyzed by a
thiolase activity.
These three activities are encoded in a multifunctional enzyme
called the mitochondrial trifunctional protein, MTP.
MTP is composed of eight protein subunits, four -subunits
encoded by the HADHA gene and four -subunits encoded by the
HADHB gene.
The -subunits contain the enoyl-CoA hydratase and long-chain
hydroxyacyl-CoA dehydrogenase activities, while the -subunits
possess the 3-ketoacyl-CoA thiolase (-ketothiolase or just
thiolase) activity.
The mammalian genome actually encodes five distinct enzymes
with thiolase activity.

Synthesis of Triglycerides
Fatty acids are stored for future use as triacylglycerols (TAGs) in all
cells, but primarily in adipocytes of adipose tissue.
TAGs constitute molecules of glycerol to which three fatty acids have
been esterified.
The fatty acids present in TAGs are predominantly saturated.
The major building block for the synthesis of TAGs, in tissues other than
adipose tissue, is glycerol.
Adipocytes lack glycerol kinase, therefore, dihydroxyacetone phosphate
(DHAP), produced during glycolysis, is the precursor for TAG synthesis
in adipose tissue.
This means that adipocytes must have glucose to oxidize in order to store
fatty acids in the form of TAGs. DHAP can also serve as a backbone
precursor for TAG synthesis in tissues other than adipose, but does so to
a much lesser extent than glycerol.

 The glycerol backbone of TAGs is activated by phosphorylation at

the C-3 position by glycerol kinase.
The utilization of DHAP for the backbone is carried out through
either of two pathways depending upon whether the synthesis of
triglycerides is carried out in the mitochondria and ER or the ER
and the peroxisomes.
In the former case the action of glycerol-3-phosphate
dehydrogenase, a reaction that requires NADH (the same reaction
as that used in the glycerol-phosphate shuttle), converts DHAP to
glycerol-3-phosphate. Glycerol-3-phosphate acyltransferase (GPAT)
then esterifies a fatty acid to glycerol-3-phosphate generating the
monoacylglycerol phosphate structure called lysophosphatidic acid.
The expression of the GPAT gene is under the influence of the
transcription factor ChREBP as described above.
The second reaction pathway utilizes the peroxisomal enzyme
DHAP acyltransferase to fatty acylate DHAP to acyl-DHAP which
is then reduced by the NADPH-requiring enzyme acyl-DHAP
reductase.

 An interesting feature of the latter pathway is that DHAP

acyltransferase is one of only a few enzymes that are targeted
to the peroxisomes through the recognition of a peroxisome
targeting sequence 2 (PTS2) motif in the enzyme.
Most peroxisomal enzymes contain a PTS1 motif. For more
information on peroxisome enzymes see the Zellweger
syndrome page.
The fatty acids incorporated into TAGs are activated to acylCoAs through the action of acyl-CoA synthetases. Two
molecules of acyl-CoA are esterified to glycerol-3-phosphate
to yield 1,2-diacylglycerol phosphate (commonly identified
as phosphatidic acid).
The phosphate is then removed, by phosphatidic acid
phosphatase (PAP1), to yield 1,2-diacylglycerol, the
substrate for addition of the third fatty acid. Intestinal
monoacylglycerols, derived from the hydrolysis of dietary
fats, can also serve as substrates for the synthesis of 1,2diacylglycerols.

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