Pyridoxine-dependent epilepsy is a rare disease that is characterized by recurring seizures that can begin in utero, at birth, or during infancy (Gospe, 1994, Goto, 2001). The seizures do not respond to standard anticonvulsants; they only stop after administration of pyridoxal-5-phosphate (PLP) (Gospe, 1994). Many patients with this particular type of epilepsy also suffer from encephalopathy (swelling of the brain), slow development, and mental retardation (Goto, 2001, Baumeister, 1994). No diagnostic tests are available for pyridoxine-dependent epilepsy (Baxter, 2001). The only method of diagnosing pyridoxine-dependent epilepsy is to observe the cessation of seizures (within minutes) upon administration of pyridoxal phosphate, and the relapse of seizures when pyridoxal phosphate is no longer available (Baumeister, 1994). The first case of pyridoxine-dependent epilepsy was documented in 1954. Since then, there have been less than 100 reported cases (Gospe, 1994). With so few cases, the disease mechanisms are still in question.
Seizures result from continued excitation of nerve cells, or neurons (Goto, 2001). Neurons transport information to and from the brain via electrical impulses called action potentials. When two neurons meet, they are separated by a small gap 20-50 nanometers wide called the synaptic cleft (Karp, 1999). The action potential from the presynaptic neuron must cross the synaptic cleft to the postsynaptic neuron via neurotransmitters (Figure 1). Neurotransmitters are chemicals that bind to receptors on the postsynaptic neuron and either excite the neuron and allow the action potential to continue, or inhibit the neuron by preventing the signal from continuing (Karp, 1999). A very important inhibitory neurotransmitter is gamma-aminobutyric acid (GABA), and when it is not present, the neurons continue to fire, and this leads to epileptic seizures. Pyridoxal phosphate plays an important role in the synthesis of GABA (Nelson, Cox, 2000).
Pyridoxal phosphate is the active form of pyridoxine (vitamin B6). It is the cofactor for a group of enzymes known as aminotransferases. Their combined purpose is to metabolize molecules that contain amino groups, especially amino acids. In the synthesis of GABA, pyridoxal phosphate binds to glutamic acid decarboxylase (GAD) and catalyzes the decarboxylation of glutamate to form GABA (Figure 2).
Pyridoxal phosphate binds to the active site of the enzyme via a Schiff-base interaction with a lysine residue (Figure 3). The presence of pyridoxal phosphate stabilizes the carbanion intermediate that is formed during the removal of the carboxyl group (Nelson, Cox, 2000).
According to a proposed mechanism for the cause of pyridoxine-dependent epilepsy, there is a low binding affinity for pyridoxal phosphate with glutamic acid decarboxylase due to a mutation in GAD (Goto, 2001). Glutamic acid decarboxylase is the primary enzyme that converts glutamate to GABA, so if pyridoxal phosphate does not bind correctly to GAD, then the enzyme will not be activated, GABA will not be synthesized, and there will be an abundance of glutamate (Goto, 2001). Glutamate is an excitatory neurotransmitter. Its presence will continue to stimulate the neurons that would normally be inhibited by GABA, thus causing seizures. The imbalance between the concentration of glutamate and the concentration of GABA is what leads to pyridoxine-dependent epilepsy (Goto, 2001). Gospe, et al., performed a study in 1994 with a patient suffering from pyridoxine-dependent epilepsy. She was diagnosed with the disease at three months of age. When pyridoxal phosphate was administered, her seizures stopped in five minutes. Her EEG (electroencephalogram), a measure of the electrical activity in the brain, had previously shown increased activity. PLP stabilized the EEG in seven minutes, which demonstrates the rapid effect of this vitamin. To confirm the proposed mechanism that binding affinity of glutamic acid decarboxylase and pyridoxal phosphate is low, the pyridoxal phosphate and GABA levels were tested in the skin fibroblasts of the patient with pyridoxine-dependent epilepsy, and compared against five controls. In the controls, no pyridoxal phosphate was administered, and the GABA levels were normal. In the patient, when no pyridoxal phosphate was administered, the GABA synthesis was much lower. However, upon administration of pyridoxal phosphate, GABA synthesis increased in all the fibroblasts, but it increased much more in the controls. This study supported the hypothesis that a defect exists within the glutamic acid decarboxylase. Since pyridoxal phosphate is important in other physiological processes, and those processes are unaffected in a patient with pyridoxine-dependent epilepsy, the mutation must occur in GAD.
Patients with pyridoxine-dependent epilepsy usually exhibit low levels of GABA and high levels of glutamate in their cerebrospinal fluid. However, one case studied by Goto (2001) included a five-month-old patient with pyridoxine-dependent epilepsy. The patient was underdeveloped and seizures were occurring every two to three days. They performed a spinal tap on the patient at three different times to measure the concentrations of glutamate and GABA: first during a seizure, second during a period of no seizures with a daily dose of pyridoxal phosphate, and third immediately preceding a seizure. The levels of glutamate and GABA were measured by HPLC. For all trials, the glutamate levels were not detectable, and the GABA levels were normal. The glutamic acid decarboxylase activity was not reduced in the patient, but she still only responded to pyridoxal phosphate. These results suggest that there is another mechanism that can cause pyridoxine-dependent epilepsy. Perhaps pyridoxal-phosphate acts as an inhibitory neurotransmitter. There is currently a theory that two forms of pyridoxine-dependent epilepsy exist. It is hypothesized that one form of the disease affects levels of GABA in the brain and spinal chord, and another form affects the levels of GABA in the brain only. This theory would explains how normal GABA levels could exist in the cerebrospinal fluid during pyridoxine-dependent seizures. However, cases such as the above-mentioned are rare, and the theory of two disease forms is very controversial.
Very small amounts of pyridoxine are needed to control the seizures (Grillo, 2001). Upon administration of pyridoxal phosphate, seizures tend to cease within minutes (Goto, 2001). Grillo (2001) found that a daily dose of 0.5mg of the pyridoxal phosphate in a multivitamin pill was sufficient to control the seizures. Unfortunately, daily vitamins containing vitamin B6 may actually hide a pyridoxine-dependent condition by suppressing the seizures, which means that the disease would not be detected. A study by Baumeister (1994) suggests that although this small amount of pyridoxal phosphate affects the seizures, it is not enough to control the disease. After measuring the glutamate level in the cerebrospinal fluid of a patient with pyridoxine-dependent epilepsy coupled with encephalopathy, they found that the amount of glutamate had increased 200 times during the seizures. They also found that level of glutamate was normalized by administration of pyridoxal phosphate. A dose of 5mg/kg body weight/day of pyridoxal phosphate controlled the seizures, but it still left the glutamate levels in the cerebrospinal fluid at a ten-fold excess. A high concentration of glutamate is neurotoxic, and results in the destruction of nerve cells, which can lead to encephalopathy (swelling of the brain). Excess glutamate levels may be the cause of the mental retardation associated with pyridoxine-dependent epilepsy. By increasing the pyridoxal phosphate dose to 10mg/kg BW/day, they were able to normalize the glutamate levels. In this particular case, the child developed normally with a higher dose of vitamin B6, and he no longer had encephalopathy. This increased amount of pyridoxal phosphate may prevent mental retardation (Baumeister, et al., 1994). In another case studied by Baxter (2001), a ten-year-old child, who had been taking 50mg/day of pyridoxal phosphate for his entire life, was failing in school. When his dosage was increased to 150 mg, his IQ, which had been below normal, sharply increased to average.
In most cases, pyridoxine-dependent epilepsy causes mental retardation even if this disease is caught early (Burd, 2000, Baumeister, 1994). For example, Goto (2001) performed a follow-up study on a boy with pyridoxine-dependent epilepsy and found that he suffered from seizures and mental retardation. Pyridoxine-dependent epilepsy is an autosomal recessive disease, and it is hypothesized that the gene that causes this condition also causes neural damage. Those with pyridoxine-dependent epilepsy may also have other seizure types accompanied with the disease that are responsive to anticonvulsants (Burd, 2000).
There are two known isomers of glutamic acid decarboxylase: GAD65 and GAD67. In studies with mice, those lacking the gene that codes for GAD65 had a greater occurrence of seizures (Goto, 2001). The GAD65 isomer was hypothesized to have a mutation resulting in a very low binding affinity of GAD to PLP. In a study by Kure (1998), it was found that in a repeat polymorphism of the GAD65 gene, two different alleles were passed to two affected offspring (siblings) maternally. These mutations, however, were present also in the controls, and are simply polymorphisms that happen to be found in both isomers. They concluded that there is another mechanism that causes pyridoxine-dependent epilepsy other than a defective glutamic acid decarboxylase enzyme.
Treatment of pyridoxine-dependent epilepsy is by lifelong daily intake of pyridoxal phosphate. However, there are adverse side effects to taking too much pyridoxine. Toxicity can occur when the dosage is high (one to two grams a day when taken by those who do not have pyridoxine-dependency) (Burd, 2000). Some of the effects are reduced spermatogenesis, clumsiness, numbness, and slower reflexes (Burd, 2000, Baxter, 2001). If there is too much pyridoxal phosphate in the body of an otherwise healthy individual, then it will continually activate the glutamic acid decarboxylase enzyme, which will produce too much GABA, and the neurons will be in a constant state of inhibition. The amount of pyridoxal phosphate prescribed for a person with pyridoxine-dependent epilepsy can only be determined by monitoring the glutamate concentration in the cerebrospinal fluid, and then adjusting the amount of vitamin B6 administered. It is now recommended that any young children suffering from seizures be tested for vitamin B6 dependency if they do not respond well to anticonvulsants (Baumeister, 1994).
There are many questions surrounding pyridoxine-dependent epilepsy that have yet to be answered. It has been hypothesized that the binding affinity of pyridoxal-phosphate to GAD is abnormally low in patients with pyridoxine-dependent epilepsy (Goto, 2001) because no other metabolic processes that rely on pyridoxine seem to be affected (Gospe, 1994). However, it has not been determined why the binding affinity between these two molecules is low. An overabundance of PLP drives the reaction via mass action and corrects the deficiency, but a mutation in the glutamic acid decarboxylase enzyme has not been discovered, and it has been suggested by Kure (1998) that the problem does not even reside in GAD. The debate is still out regarding the possibility of two forms of this disease. Finally, there is much research currently underway to find a gene that codes for the synthesis of pyridoxal-phosphate (PLP) in hopes that by obtaining more information about PLP, it will be possible to treat this disease before it starts.
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