VLCFA

April 7th, 2016 |

Stephan Kemp, Ph.D. and Paul Watkins, M.D., Ph.D.

From a biochemical standpoint, what is the problem in ALD/AMN?

In patients with ALD and AMN, there is too much Very Long Chain Fatty Acids (VLCFA) build up in most tissues of the body. This excess is most severe in the brain and the adrenal glands and it results in neurologic problems and adrenal gland malfunction (Addison’s Disease). VLCFAs also accumulate in the blood plasma. This makes it possible to diagnose ALD by a blood test.

Normal levels of VLCFA in a female suspected to have ALD. What does this mean ?

The plasma VLCFA analysis is the most frequently used diagnostic test for ALD. The assay depends upon demonstration of increased levels of C26:0 and increases in the C26:0/C22:0 and C24:0/C22:0 ratios. The experience with this assay in more than 2,000 ALD patients (males and females) has demonstrated that approximately 10 to 20% of women with ALD have normal levels of VLCFA.

A normal plasma VLCFA level thus does not exclude the diagnosis ALD. When a female is suspected to be a possible carrier for ALD, mutation analysis is the most reliable method for the diagnosis. Ideally, the mutation in the family has to be defined in an affected male or obligate heterozygote relative (an obligate carrier is a woman either with a father diagnosed with ALD or a woman with two children with genetically proven ALD).

What are fatty Acids?

Before the chemical structure of fatty acids can be explained, a little chemistry background is needed.

Most chemicals that we are familiar with, like salt and water, are made up of different building blocks called “elements”. Salt is made up of the elements sodium (Na) and chlorine (Cl). Its chemical structure is very simple: one atom or molecule of sodium is linked to one atom of chlorine. The force that holds them together is called a chemical bond. Sodium can only bond with one other molecule; the same is true for chlorine. Therefore, the chemical structure of salt can be written as Na-Cl. The line connecting the Na and the Cl represents the chemical bond.

Water is a bit more complicated. It is made up of the elements hydrogen (H), and oxygen (O). Hydrogen is like Na and Cl in that it can only “bond” with one other element. Oxygen can form two bonds. In water, two H’s bond with one O. Thus, we can write the structure of water as H-O-H. The fact that elements can bond with other elements has allowed nature to produce an incredible, almost limitless number of chemicals which make up us and our surroundings.

Fatty acids are made up of H, O, and carbon (C). Carbon can form four bonds; this makes carbon a very versatile element. The fatty part of fatty acids is a chain of carbon atoms bonded together; each C is also bonded to several H’s.

fatty acid backbone

The “acid” part of a fatty acid has one C, two O’s and one H.

In the acid portion, there are two lines or bonds between the C and one of the O’s. This is known as a “double bond”.

acid group

Examples of fatty acids

Acetic acid (vinegar), a short chain fatty acid; C2:0
acetic acid

Palmitic acid (found in lard and butter), is a long chain fatty acid; C16:0
palmitic acid

Two very long chain fatty acids (VLCFA’s) are called lignoceric acid; C24:0
lignoceric acid

and hexacosanoic acid; C26:0
hexacosanoic acid

A convenient abbreviation system is also shown with each fatty acid above. The number after the “C” is the number of carbon atoms in the chain, e.g. C2, C16, C24, C26, etc. The number after the colon tells us the number of double bonds in the carbon chain. In all the examples shown above, there are no double bonds between the carbon atoms; therefore, all have the designation “:0”.

Fatty acids – What good are they? Where do they come from and what does the body do with them?

Fatty acids are necessary chemicals in the body. They are found in more complex chemicals such as triglycerides, phospholipids, sphingolipids, glycolipids, and others. Triglycerides, are the main chemicals in “fat”; they are primarily a storage form. Phospholipids are part of the membranes that surround all cells in the body and the smaller structures inside the cells. Sphingolipids and glycolipids are complex chemicals that are found mainly in brain and nerve cells; gangliosides and myelin are in this category. The bottom line is that fatty acids are absolutely necessary for many normal body functions. We couldn’t live without them.

Fatty acids are found in the foods we eat. They are in particularly high amounts in fatty or greasy foods, fried foods, and oils. They are also present in large amounts in nuts and seeds. Meat, even lean cuts, have a lot of fatty acid in them. On the other hand, vegetables, fruits (except for the skin of the fruit), and starchy foods (e.g. pasta or breads) are relatively low in fatty acids.

What happens to dietary fatty acids? Fatty acids and other nutrients in food reach the stomach where the process of digestion begins. In this process, foods are broken down into their different components, such as carbohydrates (sugars and starches), proteins, and lipids (fatty acids and cholesterol). These components of food are then absorbed by the cells that line the intestines. Nutrients pass through the intestinal cells and into small blood vessels. These blood vessels go directly to the liver, which can be thought of as a “processing plant” for nutrients. In the liver cells, nutrients are metabolized; this means that they are either broken down to produce heat or energy, or are converted to other chemicals that the body needs. Excess nutrients can be converted to a storage form such as glycogen (for sugars) or triglycerides (for fatty acids).

Very often the body has too much fatty acid around, either because we ate too much or because the body produced too much internally. We need to get rid of the excess fatty acid. Fatty acids are then broken down or “oxidized” to produce energy or heat for the body. In fact, fatty acids are the main source of fuel for the body during starvation. There is a delicate balance between having enough fatty acid around and having too much. The body normally has finely tuned mechanisms for maintaining this balance. When the balance is shifted, disease often results.

VLCFA’s What do we know about them?

1) Are they normally present in the body?
– YES!

2) What is their function?
– They are part of brain membranes, including myelin, the “insulation” around nerve fibers.

3) Where do they come from?
– Dietary sources and through production in the body, by elongation of shorter fatty acids to longer fatty acids.

4) What could cause the increase levels of VLCFA in ALD/AMN?
– The body makes too much
– The body doesn’t remove excess amounts

5) Which of these possibilities is correct?
– Studies with patient volunteers and in cells in the laboratory have shown that the process that normally breaks down or oxidizes VLCFAs is defective in ALD/AMN.

How does the body normally oxidize fatty acids?

Through a series of chemical reactions, the body shortens fatty acids by removing two carbons at a time:

Fatty acid beta-oxidation

Palmitic acid
A fatty acid is chemically not very reactive.

The enzym fatty acyl-CoA synthetase associates a Coenzyme A to the fatty acid.
Acyl CoA
(“activated fatty acid)

The enzym acyl-CoA oxidase inserts a “double bond”
Enoyl CoA

The enzym enoyl-CoA hydratase uses a water molecule to remove the double bond and to insert a hydroxyl (O-H)
L-hydroxyacyl CoA

The “O-H” is oxidized to “=O” by the enzym hydroxyacyl-CoA dehydrogenase
Ketoacyl-CoA

The 16-carbon fatty acid is cleaved by thiolase in a 14-carbon fatty acid and a 2-carbon fatty acid.
myrestyl_CoA.jpg (13672 bytes) + Acetyl CoA

This 14-carbon fatty acyl-CoA can be further shortened by repeating the above process.
The 2- carbon unit can be used for energy production

How do enzymes work?

Working mechanism of an enzyme

Enzymes are proteins in the body that help make chemical reactions go. Enzymes have “binding sites” for the chemicals they interact with. In this illustration, the first chemical reaction of fatty acid oxidation is shown. The enzyme has one binding site for a fatty acid and another for a chemical called coenzyme A (CoA). These binding sites are very specific; fatty acid and CoA fit into them like keys in locks. By bringing these two chemical compounds close to each other, the enzyme allows them to bond together, forming a new chemical called fatty acyl-CoA. The enzyme releases this product, and is then free to bind another fatty acid and CoA. Without the enzyme, the likelihood of the fatty acid and the CoA bonding together is almost zero.

What is the “ALD protein” and what does it do?

ALDP is a transporter for VLCFA

Cells contain smaller vesicular structures called organelles. This biologic compartmentation allows the body to put enzymes that need to work together in the same place. Fatty acid oxidation takes place inside two different organelles – the mitochondria and peroxisomes. The enzymes needed for fatty acid oxidation in mitochondria are different from those in peroxisomes. Research done at the Kennedy Krieger Institute in the 1980’s by Drs. Inderjit Singh and Hugo Moser showed that VLCFA oxidation takes place in peroxisomes only (Singh et al 1981).

In 1993, Drs. Patrick Aubourg and Jean-Louis Mandel in Paris discovered that the ALD gene generates a protein (ALDP) that is localized inside cells in the membrane of the peroxisomes (Mosser et al 1993). The ALD protein belongs to a specific family of transporter proteins. These proteins allow molecules to cross biological membranes like those surrounding the cellular organelles.

ALD is characterized by the inability of cells to metabolize/degrade VLCFA to shorter-chain fatty acids. This results in elevated VLCFA levels in all tissues of the body. Degradation of VLCFA takes place exclusively in peroxisomes. The enzymes that are required for the breakdown of VLCFA are functional and present inside the peroxisomes in ALD patients. Based on studies demonstrating that the expression of normal ALD protein in ALD patient cells restored VLCFA beta-oxidation (Shinnoh et al 1995) and reduced VLCFA to normal levels (Cartier et al 1995), it has long been hypothesized that ALDP transports VLCFA across the peroxisomal membrane. Experiments using yeast cells and cells from ALD patients provided evidence that ALDP indeed transports VLCFA (as VLCFA-CoA) across the peroxisomal membrane (van Roermund et al 2008; Ofman et al 2010).

The ALDP deficiency in ALD has two major consequences: 1) it impairs peroxisomal VLCFA beta-oxidation and 2) it raises VLCFA-CoA levels in the cytosol of the cell. These elevated levels of VLCFA-CoA in the cytosol are substrate for further elongation to even longer fatty acids by ELOVL1, the human C26-specific elongase (Ofman et al 2010; Kemp and Wanders 2010).

ALDP

The ALD protein (ALDP) is not detectable by means of immunofluorescence analysis in about 70% of affected individuals. For reasons that are not well understood, the gene product may be absent even in individuals who have missense mutations. The principle biochemical abnormality is the accumulation of saturated very long-chain fatty acids, particularly hexacosanoic (C26:0) and tetracosanoic (C24:0) fatty acids, as a result of the impaired capacity to degrade these substances, a function that normally takes place in the peroxisome. The ALDP protein transports VLCFA from the cytosol to the peroxisome.


Figure: Detection of ALDP using immunofluorescence: (left) fibroblasts from a control show punctate staining indicating the normal presence of ALDP in peroxisomes; (middle) fibroblasts from a male patient with ALD and a mutation that affects ALDP stability. Note that there is no punctate staining; (right) fibroblasts from a female patient with ALD from the same family as the male patient. The ABCD1 gene is located on the X-chromosome. Females have two X chromosomes. However, in each cell only 1 X-chromosome is active. The cells that show punctate staining are those that have an active copy of the normal ABCD1 gene, while those that do not show punctate staining are the cells that have an active ABCD1 gene harboring the mutation.
Images were made by Dr. Merel Ebberink

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