SBK3013 : PRINCIPLES OF BIOCHEMISTRY

Carnitine Deficiencies

-FROM LEFT..

#MAHIRAH#JIAH#ATIQAH#MEERA#BELLA#ZAKIAH

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?• Dicuss on the significance presence of lipid vacuole in muscle

biopsy• Explain Mitochondrial (beta) β-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 boy’s 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

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 long-chain 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.

Role of Carnitine?

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)

• 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.

• 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.

Primary Carnitine Deficiency Secondary Carnitine Deficiency

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 β-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. Acetyl-L-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 they’re 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 phosphateGlycolysis (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 therapiesDevelopment 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 lactateb) pyruvate enters the Krebs Cycle in mitochondriac) 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 fiberscan be demonstrated by PAS stain in glycogenoses. Similarly, lipid accumulation isDemonstrated 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) β-Oxidation Reactions

The process of mitochondrial fatty acid oxidation is termed β-oxidation since it occurs through the sequential removal of 2-carbon units by oxidation at the β-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 β-oxidation.

Each round of β-oxidation involves four steps that, in order, are oxidation, hydration, oxidation, and cleavage.

The first oxidation step in mitochondrial β-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 4–6 carbons in length; medium-chain acyl-CoA dehydrogenase (MCAD) prefers fats of 4–16 carbons in length with maximal activity for C10 acyl-CoAs; long-chain acyl-CoA dehydrogenase (LCAD) prefers fats of 6–16 carbons in length with maximal activity for C12 acyl-CoAs.

The next three steps in mitochondrial β-oxidation involve a hydration step, another oxidation step, and finally a hydrolytic reaction that requires CoA and releases acetyl-CoA 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 NAD-dependent long-chain hydroxacyl-CoA dehydrogenase activity (3-hydroxyacyl-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 α-subunits encoded by the HADHA gene and four β-subunits encoded by the HADHB gene.

The α-subunits contain the enoyl-CoA hydratase and long-chain hydroxyacyl-CoA dehydrogenase activities, while the β-subunits possess the 3-ketoacyl-CoA thiolase (β-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 acyl-CoAs 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 1,2-diacylglycerols.

THANK YOU