Peroxisomes are single membrane bound cellular organelles present in all mammalian cells except erythrocytes. In mammals, they are abundant in the liver and kidney. These small multi-functional organelles are involved in a series of vital cellular reactions such as free radical detoxification, α and β oxidations of fatty acids, and biosynthesis of bile acids, DHA, and ether lipids. It has been suggested that peroxisomes vary between different organisms in their enzymatic content, size, and number (₄). Peroxisomal proteins (called peroxins) are encoded by nuclear genes (called PEX-genes) and are synthesized by cytosolic ribosomes. The synthesized proteins are transported into peroxisomes. This indicates that there must be a transport system that facilitates the transportation of newly synthesized proteins into peroxisomes (₃). Up to date, 15 human PEX genes have been identified.

Branched-chain fatty acids, such as Phytanoyl-CoA, cannot undergo β oxidation due to the presence of a methyl group at the β carbon of fatty acids (see figure 1). However, peroxisomes have the necessary enzymes to make a new unmethylated β carbon, through α oxidation, to facilitate the subsequent β oxidation process. Milk and animal fats are rich in Phytanoyl-CoA.

Figure 1

Figure 1: and Oxidations of Fatty Acids

Mitochondria of animal cells are the major sites for β oxidation of fatty acids. However, peroxisomes of animal cells, similarly to mitochondria, carry out the four fundamental steps of β oxidations, namely: initial dehydrogenation, hydration, dehydrogenation, and thiolytic cleavage. Despite this similarity, there are three major differences regarding the oxidative mechanism of fatty acids in these two organelles of animal cells:

1. The initial dehydrogenation reaction of the β oxidation is different in mitochondria and peroxisomes. Mitochondrion uses its initial dehydrogenation step to oxidize fatty acids to generate electron carrier coenzyme, FADH₂ (reduced Flavin Adenine Dinucleotide) from FAD (Flavin adenine dinucleotide). FADH₂ carries electrons which, in the subsequent steps, are funneled into the unique oxidative phosphorylation pathway to regenerate FAD and to produce ATP and water. Similarly, peroxisomes generate FADH₂. However, as a consequence to the lack of the oxidative phosphorylation pathway, peroxisomes synthesize a toxic molecule, hydrogen peroxide (H₂O₂, see figure 2). Fortunately, due to the presence of peroxisomal catalase enzyme, hydrogen peroxide is immediately broken down into harmless water and oxygen before it exerts any damage to the cell.

Peroxisomes are more active than mitochondria in oxidizing VLCFA (ie., fatty acids with more than 20 carbons) such as hexacosanoic acid and branched-chained fatty acids such as phytanic acid and pristanic acid from diary products. Phytanic acid has a methyl group on its β carbon which renders the β oxidation process. However, as it was explained in the introduction, following the α oxidation, the β oxidation facilitates phytanic acid oxidative metabolic pathway. Pristanic acid, which is produced upon α oxidation of phytanic acid, and phytanic acid are used as energy sources and are oxidized to produce ATP.

3. Due to the lack of citric acid cycle enzymes in the peroxisomes, acetyl-CoA, the end product of β oxidation, is not further oxidized in peroxisomes, rather is exported into the cytoplasm. Therefore, many of the VLCFA or branched-chain fatty acids are metabolized to shorter-chain fatty acids, such as hexanoyl-CoA, instead of completely being oxidized to acetyl-CoA. The shortened fatty acids are exported to mitochondria to be completely oxidized to provide energy in the form of ATP.

Figure 2

Figure 2: Oxidation of Fatty Acids in the Mitochondria and Peroxisomes of animal cells.

Ether lipids, including plasmalogens and platelet-activating factor, are important phospholipids which differ from other fatty acids by having an ether group (ether- linkage) instead of an ester group (see figure 3). Plasmalogens play important role in maintaining the integrity of cell membranes. They are found in all mammalian cells with high amount in muscle, heart, and brain. A significant fraction (about 50%) of heart tissue's phospholipids in vertebrate is enriched in plasmalogens. Plasmalogens play important role in the neural tissues too. Consequently, when the synthesis of plasmalogens is impaired, muscle, heart and brain tissues are affected. Platelet-activating factor is a potent molecular signal that is released from basophils. This factor is involved in acute inflammation, allergic response, platelet aggregation, and the release of the neurotransmitter serotonin from platelets. Serotonin activates platelet aggregation which plays an important role in the blood clotting process (₅).

Figure 3

Figure 3: The structure of plasmalogens, a typical ether lipid.

Liver peroxisomes play an important role in the biosynthesis of bile acids (₆). Bile acids are derivatives of cholesterol which are synthesized in the liver. Synthesis of bile acids represents the major route for elimination of cholesterol from the body. Approximately 50% of cholesterol is metabolized into cholic acid and chenodeoxycholic acid (see figure 4). These amphipathic molecules are transported and stored in the gall bladder. After ingestion of fatty meals, bile acids are released into small intestine in order to facilitate lipid digestion.

Humans have a total of four different bile acids with 24 carbon atoms in each. The amphipathic property of these molecules permits them to act as detergents assisting emulsification of ingested lipids into soluble micelles. This in turn facilitates the enzymatic digestion and absorption of fats. Lipids are the major source of energy (ATP). ATP plays a major role in the contraction of muscle which involves both myosin and actin proteins (₇). A lack of fat absorption and ATP formation correlates well with the “floppiness” of muscles tone (see the case).

Figure 4

Figure 4: The structure of chenodeoxycholic acid and cholic acid, two primary bile acids.

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid with 22 carbon atoms and six cis double bonds (22:6, see figure 5). DHA is found in diet, particularly fish, or is synthesized in the peroxisomes from linolenic acid. Because DHA's last double bond (the first double bond from the omega carbon) mimics the last double bond of linolenic acid, it belongs to the omega-3 group fatty acids (in contrast to linolenic acid that refers to short-chain omega-3 fatty acid, DHA refers to long-chain omega-3 fatty acid) (₈). DHA is a major fatty acid in the sperm, brain, and retina.

Figure 5

Figure 5: Docosahexaenoic acid (DHA)

Many enzymes are unstable in the cellular peroxisomes in patients with ZS (₁₃). As a result, many of the peroxisomal metabolic functions are absent. However, it is not yet clear how exactly abnormalities in peroxisomal metabolic pathways lead to ZS. Several hypotheses have been made from different studies to explain a link between peroxisomal deficiencies and ZS. In a developing fetus for a few weeks after birth, peroxisomes are abundant in the oligodendrocytes, the cells which surround the developing central nervous system. The excess of VLCFA, due to the lack of oxidation process in peroxisomes, has been suggested to interfere with the growing axons and results in impairment of neuronal migration (₁₄). Plasmalogens are important molecules in the structure of myelin. Animal model studies have shown that the reduced levels of plasmalogens and platelet activating factor in ZS patients is associated with abnormality in neuronal migration (₁₅). Interestingly, a defect in catalase, an important enzyme of peroxisomes, has not been associated with ZS. This indicates that the toxicity of H₂O₂ is not a cause for the ZS.

The synthesis of platelet-activating factor is reduced in patients with ZS which results in an impaired blood clotting process. The affected children are given vitamin K and blood clotting factors to facilitate the blood clotting process.

As it was mentioned earlier, peroxisomes contain enzymes that carry out β oxidation of VLCFA. If VLCFA are not oxidized in peroxisomes, they will accumulate in glial cells including oligodendrocytes. The unprocessed VLCFAs are incorporated into glial cell membranes which results in the cell dysfunction and cell death (₁₆). In addition, the accumulation and incorporation of VLCFA in the myelin causes demolition of myelin resulting in hypomyelination. Myelin's function is to insulate the axon through which the nerve impulses are conducted.

Dietary fats provide two essential fatty acids, linoleic acid and linolenic acid, which humans need for the synthesis of eicosanoids (prostaglandins, thromboxanes, and leukotrienes). Because bile acids facilitate digestion and absorption of lipids in the small intestine, deficiency in the bile acids synthesis results in malabsorption of lipids, including the essential fatty acids, in the ZS patients. Clinical trials are underway to determine the effectiveness of oral bile acid therapy, including cholic acid, in patients with ZS (₁₇).

It has been reported that deficiency in the synthesis of DHA leads to abnormal development of the brain and retina because, in the experimental studies, low levels of DHA have been associated with severe visual and neurological damage (₁₈).

Clinical manifestations in ZS are due to a range of severely affected organs such as liver, eye, cartilage, heart, muscle, and brain. Typical and apparent diagnostic prototypes are: high forehead and flattened face; broad nose and widely spaced eyes, respiratory distress and bell-shaped chest (₁₂); severe hypotonia or “floppiness” of muscles tone and an inability to move, glaucoma, difficulty to suck and/or swallow; hyporeflexia; epileptic seizures, nystagmus, mental retardation, impaired hearing, and enlarged liver. These clinical symptoms are important to know in order to diagnose ZS accurately.

There are a few markers that screen functional peroxisomes. Because peroxisomes are the sites for oxidation of VLCFA and branched-chain fatty acids, increased level of these two molecules are often used as diagnostic tools for the manifestation of ZS. An increased plasma level of VLCFA is an indication of ZS. Prenatal analysis for VLCFA and plasmalogen are examples that are used from amniotic tests. Absence of peroxisomes in liver biopsy is another sign of ZS. In addition, a high amount of arachidonic acids metabolites may excrete into the urine of ZS patients (₁₉).

Docosahexaenoic acid (DHA)

Actigall

Zantac

Pulmicort Respules

Pregestimil

Phenobarbital and Tegretol

Vitamin A, D, E, and K

ZS is an autosomal recessive disorder which means there is always a probability that a single genetic defect can lead to the devastating symptoms mentioned in the case. So far only a few genetic diseases can be treated. Genetic counseling is important to inform parents of the severity of ZS. Genetic tests can enable parents to make more informed decision. It is important for parents to understand the absolute and relative risks that a new born baby will inherit ZS. Of particular concern are families who have (had) at least one child with ZS. Not only it is important to diagnose a child but it is important to diagnose the parents to predict the probability of having a child with ZS.

Pharmacists provide extensive information regarding appropriate medication use. They confirm indication and dosing, provide guidance on potential side effects, cautions or monitoring parameters. In this case, phenobarbital dosing must be adjusted in cases of renal impairment. Additionally, some medications (such as phenobarbital) are available in solution forms, which would make it easier for ZS parents to administer to their children via feeding tubes.

Pharmacists also are the health care professionals patients see on a regular basis, up to 10-12 times per year. As a result, a relationship of trust often develops. It is important for pharmacists to understand how difficult it is for parents knowing their child would not live to see her/his first birthday. When patients are dealing with difficult health concerns, they often seek the pharmacist's interpretation. In situations of genetic predispositions, patients can discuss information obtained from the genetic counselors with their pharmacist. Pharmacists can offer additional information or perspective.

A very special note of gratitude to Grace, Samuel, and hundreds of other affected children with Zellweger Syndrome.