**1. Introduction**

Vitamin B6 (vitB6) is a generic term that refers to a group of six interconvertible chemical compounds that share a pyridine ring in their centre. These vitB6 compounds (also called vitamers) are pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL) and their 5′-phosphorylated forms pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP) and pyridoxal 5′-phosphate PLP) [1] (**Figure 1**). VitB6 is required by all living organisms for their survival, but only microorganisms and plants can carry out *de novo* synthesis of this vitamin*.* Other organisms including humans acquire vitB6 from exogenous sources and interconvert its different forms according to their needs using a biochemical pathway known as the salvage pathway [1, 3].

#### **1.1 Metabolism of vitB6**

Among the six vitB6 compounds, PLP is the biologically active and most important vitamer since it is required as a cofactor for a multitude of enzymes in the body. Humans and other mammals obtain PLP directly from diet or through synthesis from other vitameric forms ingested with food or recycled from degraded PLP-dependent enzymes via the salvage pathway [1, 4] (**Figure 2**). The central enzyme in this pathway is PNP oxidase (PNPO), a flavin mononucleotide (FMN) dependent enzyme that is capable of converting PNP or PMP to the active cofactor

#### **Figure 1.**

*Chemical structures of the six vitamin B6 vitamers. Colored atoms designate oxygen or hydroxyl group (red), nitrogen or amine group (blue) and phosphorus (brown). (Retrieved from [2]).*

*Vitamin B6 and Related Inborn Errors of Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99751*

**Figure 2.**

*The PLP salvage pathway. Phosphorylated vitamers are converted to PLP by the enzyme PNPO. PLP is also recycled from degraded holo-B6 enzymes through PMP as an intermediate step. ADP: adenosine diphosphate, ATP: adenosine triphosphate, FMNH2: flavin mononucleotide reduced form, Ph'ases: phosphatases, Pi: inorganic phosphate. (Based on [5–7]).*

PLP [1]. Other important enzymes in the salvage pathway are PL kinase (PLK) and a number of different phosphatases [5].

VitB6 vitamers are widely available in animal and plant food sources. PLP and in a lesser amount, PMP are present as such in animal-derived foods, mainly associated with muscle glycogen phosphorylase, while plant foods are more enriched in PN, PNP and PN glucosides [1, 4, 8].

After being ingested, phosphorylated vitamers (PLP, PNP and PMP) undergo dephosphorylation by the ecto-enzyme tissue-specific intestinal phosphatase (IP) [5], whereas PN glucoside (PNG) vitamers from plants are hydrolyzed by a glucosidase before absorption [1, 10]. Absorbed vitamers are carried by the portal circulation to the liver where they are phosphorylated by PLK [5]. Inside liver cells, PNP and PMP are oxidized by PNPO to form PLP, which is then released to the circulation bound to lysine-190 residue of albumin (**Figure 3**) [9–11]. Binding of PLP to albumin is thought to protect the cofactor from hydrolysis and other reactions [11]. About 60% of circulating vitB6 is in the form of albumin-bound PLP, while PN, PM and PL constitutes the remaining proportion [5].

Prior to delivering the circulating PLP to different tissues, it is dephosphorylated to PL by the ecto-enzyme tissue nonspecific alkaline phosphatase (TNSALP) to enable entry into the cells and through the blood–brain barrier. Inside the cell, PL is re-converted by PLK to PLP, which now can be used as a cofactor in many biochemical reactions (**Figure 3**) [1, 5, 9]. Degradation of PLP-bound enzymes (holo-B6 enzymes) can generate PMP, which is then oxidized back to PLP by the action of PNPO [6] (**Figures 2** and **3**).

Besides the liver, it has been shown that the intestine also contributes an important role in vitB6 metabolism. *In vitro* studies utilizing human intestinal

#### **Figure 3.**

*Metabolism of vitB6 vitamers in different tissues of the body. PNGH: PNG hydrolase; PLPase: PLP phosphatase; AOX/DH: aldehyde oxidase/dehydrogenase; BBB: blood–brain barrier; PA: pyridoxic acid; E-PLP: enzyme-bound PLP; E-PMP: enzyme-bound PMP. (Based on [6, 7]).*

epithelial Caco-2 cells [12] demonstrated that, after incubation of these cells with multiple vitB6 vitamers, PL was the only vitamer detected at the basolateral side which indicated that all other vitamers were converted to PL inside the intestinal cells. Excretion of PN and PM at the basolateral side was only detected when the enterocytes were incubated with high concentrations of these vitamers. The authors suggested that under normal dietary intakes, PN and PM are converted to PL by the enterocytes and PL becomes the principal vitamer that reaches the portal circulation. All other organs including the liver can then obtain PL from the circulation and only require PLK to produce PLP. Under high vitB6 intakes, however, the ingested amounts of PN or PM may surpass the intestine's capacity to fully metabolize these vitamers. In this case, PN and PM will be released to the

*Vitamin B6 and Related Inborn Errors of Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99751*

portal circulation and will subsequently be converted to PLP in the liver. The study also showed the expression of a full battery of the salvage enzymes in Caco-2 cells as well as in lysates of human intestine, adding further evidence for a major role of the intestine in vitB6 metabolism [12]. Earlier works in mice [13, 14] have also pointed to a similar role of the intestine. In these studies, following oral administration of radiolabeled PN, labeled PL and PLP were detected in the mouse intestine and portal circulation indicating involvement of the intestine in converting dietary vitamers to circulating PL [13, 14].

#### **1.2 Catabolism of vitamin B6**

At the other end of vitB6 metabolism, little is known about the catabolic pathways in humans or other mammals. In contrast, these mechanisms are well established in microorganisms [3, 11, 15]. In humans and other mammals, the primary product of the degradation of PLP (and all other vitB6 vitamers) is 4-pyridoxic acid (4-PA). This compound, which is excreted in urine, is generated in two steps. In the first one, PLP is hydrolyzed to PL by the action of an intracellular enzyme known as PLP phosphatase (PLPase). In the following step, PL is oxidized to 4-PA by a non-specific aldehyde oxidase (AOX) or aldehyde dehydrogenase (**Figure 3**) [3, 6, 12, 15, 16]. In microorganisms, 4-PA is further degraded to other metabolites that can be utilized by the cell in various biochemical processes [15]. Some microbial vitB6 catabolic products such as 5-pyridoxic acid (5-PA), 5-pyridoxolactone [17] and 4-pyridoxolactone [17, 18] have been also discovered in human individuals under consumption of high amounts of vitB6. Several other PN derivatives have been identified in humans and/or other mammalian species, but their biochemical pathways and precise functions have not yet been unraveled.

For example, Coburn and Mahuren [19] detected pyridoxine 3-sulfate, pyridoxal 3-sulfate and *N*-methylpyridoxine in the urine of domestic cats, and, interestingly, these chemicals were excreted at concentrations higher than 4-PA, even with moderate intake of PN. Other studies reported the discovery of multiple PM derivatives in urine samples from PM-administered diabetic and obese rats [20, 21]. Moreover, at least nine unidentified vitB6 metabolites were detected in human urine after oral administration of radiolabeled PN [17, 19].

Oxidation of PN at the 5′ position, followed by sequential dehydrogenation to form 5-PA, is known to exist only in the PN catabolic pathway of some bacterial species like *Pseudomonas* IA and *Arthrobacter* Cr-7, where the enzymes catalyzing these reactions have been characterized [15]. Similar reactions have been proposed to occur in mammals based on experimental clues. The first one was provided by the study of Coburn and colleagues [22] who showed that healthy men who ingested a structural analog of PN, 4′-deoxypyridoxine, excreted 4'-Deoxy-5-pyridoxic acid in their urine. A similar experiment was carried out in guinea pigs [23], and the results indicated that these animals were also able to convert 4′-deoxypyridoxine to 4′-deoxy-5-pyridoxic acid. All together, these studies provided evidence for the possible existence of alternative but currently undiscovered catabolic routes of PN in humans and other mammals.

#### **1.3 Vitamin B6 transportation across cellular membrane**

Multiple experimental evidence suggests that, as with most water-soluble vitamins [24], the transportation of vitB6 across mammalian cell membrane is carrier-mediated. Studies in cultured human intestinal [12, 25], colonic [26], and renal cells [27] and animal-derived renal proximal tubular cells [28] demonstrated the presence of an efficient and specific carrier-facilitated mechanism for cellular

uptake of vitB6. Such a specific transporting membrane carrier was employed to produce a high affinity gene delivery system into cancer cells using a vitB6-coupled vector [29]. However, the molecular identity of vitB6 transporter protein in mammals has remained elusive [12, 30]. Among eukaryotes, the only vitB6 transporters identified so far are the yeast transporters, Tpn1p [31] and Bsu1 [32], and, recently, PUP1 in plant species *Arabidopsis* (first to be identified in plants) [33].

### **1.4 Physiological roles of vitamin B6**

PLP, the coenzymatically active form of vitamin B6, plays an important role in maintaining the biochemical homeostasis of the body [34]. In the human body, PLP is an essential cofactor for more than 140 distinct enzymatic activities, mainly associated with synthesis, degradation and interconversion of amino acids as well as with neurotransmitter metabolism [35–38]. PLP-dependent enzymes are also involved in a multitude of other cellular processes, including biologically active amine biosynthesis, lipid metabolism, heme synthesis, nucleic acid synthesis, protein and polyamine synthesis and several other metabolic pathways (**Figure 4**) [5, 6]. Furthermore, PLP is important in energy homeostasis through glycogen degradation and gluconeogenesis, since PLP is a cofactor for glycogen phosphorylase and gluconeogenic transaminases [36, 41]. In folate-mediated one-carbon metabolism (FOCM), PLP is required as a cofactor for the enzyme serine hydroxymethyltransferase, both its cytoplasmic (SHMT1) and mitochondrial (SHMT2) isoforms. FOCM is an important pathway that is involved in a number of physiological processes such as DNA methylation, redox homeostasis and purines and thymidine biosynthesis [36, 42].

As a coenzyme for the synthesis of several neurotransmitters including D-serine, D-aspartate, L-glutamate, glycine, γ-aminobutyric acid (GABA),

#### **Figure 4.**

*The diverse cellular functions of PLP. Names in blue are the PLP-dependent enzymes involved in each metabolic process. Some enzymes can be implicated in mutiple processes. An example is branched-chain amino acid aminotransferase which can fall under amino acid and neurotransmitter metabolism. Glycine dehydrogenase can be classified under folate cycle and amino acid and neurotransmitter metabolism. (Based on [6, 39]; PLP chemical structure was retrieved from [40]).*

*Vitamin B6 and Related Inborn Errors of Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99751*

serotonin, epinephrine, norepinephrine, histamine and dopamine, PLP is an important vitamer for normal brain function [5, 43]. For example, GABA, the major inhibitory neurotransmitter in the central nervous system (CNS), is synthesized from L-glutamate by the PLP-dependent enzyme glutamate decarboxylase (GAD). Moreover, PLP is a cofactor for branched-chain amino acid aminotransferase (BCAT) which catalyzes the synthesis of L-glutamate, the major excitatory neurotransmitter, from branched-chain amino acids like leucine and valine [5].

Another important PLP-dependent enzyme in the brain is aromatic L-amino acid decarboxylase (AADC), which catalyzes the final steps in the biosynthetic pathways of serotonin and dopamine (**Figure 5**) [5, 36]. These neurotransmitters also serve as precursors for other important compounds in the brain, specifically melatonin, norepinephrine and epinephrine (**Figure 5**) [5, 46].

In addition to its role as an enzymatic cofactor, PLP has been shown to play a role in preventing DNA damage [47] and in modulating the activity and expression of steroid hormone receptors [6, 48]. vitB6 has also been described as an efficient antioxidant in plants and fungi, with the ability of its different vitamers to quench reactive oxygen species [1, 49, 50].

#### **1.5 PLP homeostasis and its importance for human health**

PLP is a highly reactive compound because of its aldehyde group at the 4′ position which can undergo spontaneous complexation with other molecules within the cell [1, 9]. It may bind with amino groups in proteins and disrupt their structure [6]. For example, it has been shown that PLP can react with the lysine residue in the

#### **Figure 5.**

*Biosynthetic pathway for biogenic amine neurotransmitters and melatonin. The PLP-dependent enzyme, AADC, catalyzes a central step in this pathway. TH: tyrosine hydroxylase; TPH: tryptophan hydroxylase; L-dopa: levodopa; 5-HTP: 5-hydroxytryptophan; D*β*H: dopamine* β*-hydroxylase; MAO: monoamine oxidase; SNA: serotonin N-acetylase; HIOMT: hydroxyindole O-methyltransferase; COMT: catechol-Omethyltransferase; ALDH: Aldehyde dehydrogenase; 3-OMD: 3-O-methyldopa; VLA: vanillactic acid; 5-HIA: 5-hydroxyindole acetaldehyde; 5-HIAA: 5-hydroxyindoleacetic acid; 3-MT: 3-methoxytyramine; HVA: homovanillic acid; DOPAC: 3,4-dihydroxyphenylacetic acid. (Based on [44, 45]).*

active site of human DNA topoisomerase I, causing its inhibition [51, 52]. Through a chemical reaction known as Knoevenagel condensation, PLP can also react with intermediate metabolites like Δ<sup>1</sup> -pyrroline 5-carboxylate and Δ<sup>1</sup> -piperideine 6-carboxylate, which form the molecular basis of PLP depletion in the neurometabolic diseases ALDH7A1 deficiency and hyperprolinaemia type II, respectively [6]. Because of its high reactivity and to prevent toxic accumulation of this cofactor, the intracellular pool of free PLP is maintained at very low concentration (about 1 μM in eukaryotic cells) [1, 5, 6]. It is therefore likely that PLP production in the cell is tightly regulated [5], and experimental work indicates the presence of an efficient mechanism that maintains intracellular PLP levels within optimum levels [12]. However, how the concentration of PLP is controlled in mammalian tissues is not entirely understood [3, 34].

A number of mechanisms have been proposed that help in PLP homeostasis. First, both enzymes that produce PLP, PLK and PNPO, are inhibited by their product PLP and its rate of synthesis can, therefore, be controlled by this feedback inhibition [1, 5, 6]. Enzymes that degrade PLP and PL, like PLPase and AOX, respectively, have also been proposed as a mechanism that keeps free PLP at low level within the cell [1, 5, 6]. Proteins that are known to naturally bind PLP, like muscle glycogen phosphorylase, plasma albumin and hemoglobin in red blood cells, contribute to reducing the amount of free reactive PLP [6]. In addition to its catalytic role in PLP synthesis, a recent study [53] demonstrated that PNPO forms a tight a binding with PLP at a noncatalytic site *in vitro*. The study further showed that PLP-bound PNPO interacts with several PLP-dependent enzymes and hypothesized that it may serve as a safe carrier of the reactive cofactor to its dependent enzymes [53]. Another more recently proposed PLP carrier protein is known as PLPHP or PLP Homeostasis Protein (*described in detail in Section 2.5*).

Conditions that disrupt cellular PLP homeostasis can cause disease. For example, inactivation of PLPP in mice led to increase in PLP levels, anxiety and motor deficits [54]. In humans, intake of high doses of vitB6 is known to cause motor and sensory neuropathies [1, 5]. Deficiency of PLP in the cell is also implicated in several pathologies, most notably the so-called vitB6-dependent epileptic encephalopathies [1, 5, 9, 37].

### **2. VitB6-dependent epileptic encephalopathies**

VitB6-dependent epileptic encephalopathies (B6EEs) represent a clinically and genetically heterogeneous group of rare inherited metabolic diseases [55, 56]. These debilitating conditions are characterized by recurrent seizures in the prenatal, neonatal, or postnatal period, which are typically resistant to conventional anticonvulsant treatment but well-controlled by the administration of PN or PLP [56–59]. In addition to seizures, children affected with B6EEs may also suffer from developmental and/or intellectual disabilities, along with structural brain abnormalities [60]. The 5 principal types of B6EEs: PN-dependent epilepsy due to ALDH7A1 (antiquitin) deficiency (PDE-ALDH7A1) (MIM: 266100), hyperprolinemia type 2 (MIM: 239500), PLP-dependent epilepsy due to PNPO deficiency (MIM: 610090), hypophosphatasia (MIM: 241500) and PLPBP deficiency (MIM: 617290) [6, 9, 60, 61] (**Table 1**). According to the underlying pathobiochemical mechanism, these forms of B6EEs can be categorized into: 1) defects in amino acid catabolic pathways causing buildup of byproducts that react with PLP (PDE-ALDH7A1 and hyperprolinemia type 2), 2) defects in the vitB6 salvage pathway (PNPO deficiency), and 3) defects in cellular uptake of PLP (hypophosphatasia) [6, 9] (**Table 1**). In the most


### *Vitamin B6 and Related Inborn Errors of Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99751*

**Table 1.**

*Summary of the genetic, biochemical and clinical features of B6EEs.*

recently discovered type, PLPBP deficiency, the exact mechanism that disrupts PLP homeostasis is not fully understood [7].

### **2.1 PN-dependent epilepsy (ALDH7A1 deficiency)**

## *2.1.1 Disease mechanism*

PN-dependent epilepsy (PDE-ALDH7A1) is caused by homozygous or compound heterozygous mutations in the *ALDH7A1* gene (also known as antiquitin, ATQ ). *ALDH7A1* codes for α-aminoadipic semialdehyde dehydrogenase, an enzyme that functions within the lysine catabolism pathway in the brain and peripheral tissues [62]. In PDE-ALDH7A1, loss of the enzyme's function leads to the accumulation of three upstream lysine catabolites: ∆<sup>1</sup> -piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid (PIP) [60] (**Figure 6**). Through a chemical reaction known as Knoevenagel condensation, accumulating P6C spontaneously conjugates with PLP, forming inactive complex products and causing cellular deficiency of this important cofactor [62] (**Figure 6**). Seizures are thought to occur because PLP is required for neurotransmitter metabolism, particularly for the synthesis of GABA from glutamate [71].

#### *2.1.2 Clinical features*

The main clinical manifestation of PDE-ALDH7A1 is recurrent perinatal-onset seizures that are resistant to conventional anticonvulsant treatment, but which show remarkable response to the administration of high doses of PN [60, 72]. Seizures usually relapse when PN treatment is discontinued, either incidentally or for diagnostic purposes [60]. In some cases, the mother of an affected child has described abnormal fetal movements during pregnancy, suggestive of pre-natal onset of seizures [55, 73–75]. In atypical cases, seizure onset can be delayed to up to 3 years of age [60], and in one exceptional case, Srinivasaraghavan et al. [76] reported an Indian female with genetically proven PDE-ALDH7A1 in whom seizures did not start until the age of 17 years (juvenile onset).

In addition to seizures, most PDE-ALDH7A1 patients (about 75%) also suffer from developmental delay and moderate to severe intellectual disability [60, 72, 77]. In addition, as revealed by neuroimaging analysis, a spectrum of structural brain defects have been described in affected children with anomalies of corpus callosum (agenesis/hypoplasia/dysplasia) and white matter being common features [75, 77–79]. Motor deficits (hypotonia/hypertonia/dystonia), irritability, autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD) and anxiety are additional features reported in patients [75, 77, 80].

The phenotypic spectrum of PDE-ALDH7A1 may also include non-neuronal features, but these are less frequently observed in patients. Reported examples are ocular problems, hypoglycemia, hypothyroidism, lactic acidosis, profound electrolyte disturbances, diabetes insipidus, coagulopathy, anemia, respiratory distress and hypotension [60, 77, 79, 81, 82].

#### *2.1.3 Biochemical features and diagnostic biomarkers*

In PDE-ALDH7A1, blockade of the ATQ-catalyzed step in the lysine catabolism pathway leads to accumulation of 3 upstream metabolites, P6C, α-AASA and PIP, as discovered by screening of patients' body fluids. Presence of these metabolites in supraphysiological levels is considered the hallmark biochemical feature of ATQ *Vitamin B6 and Related Inborn Errors of Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99751*

#### **Figure 6.**

*Pipecolic acid (left) and saccharopine (right) pathways for L-lysine catabolism in mammals. The two pathways converge at the step of* α*-AASA/P6C synthesis. ALDH7A1 catalyzes the step indicated by the red "X". Inactivation of the enzyme in PDE-ALDH7A1 causes buildup of its two substrates, P6C and* α*-AASA, as well as of PIP (the 3 biomarkers in patients). Accumulating P6C condenses with PLP, forming an inactive product and leading to depletion of the cofactor. \*The nature of the first step of pipecolic acid pathway is undetermined. AASS: aminoadipic semialdehyde synthase; LKR: lysine-ketoglutarate reductase; SDH: saccharopine dehydrogenase; AADAT: 2-aminoadipate aminotransferase; KR: ketimine reductase; CRYM: Mu-crystallin homolog; PIPOX: pipecolic acid oxidase; P5CR: piperideine-5-carboxilic reductase. (Based on [69, 70]).*

deficiency and have been utilized as diagnostic biomarkers [72]. Recently, two additional lysine metabolites discovered to accumulate in patients have been suggested as novel biomarkers. The first one is 6-oxopipecolate (6-oxo-PIP), which was found to be present in large concentrations in plasma, urine, and CSF of ATQ deficiency patients [83, 84]. By means of an untargeted metabolomics approach, Engelke et al. [83] identified another novel metabolite, 6-(2-oxopropyl)piperidine-2-carboxylic acid (2-OPP), that accumulated in biofluids of affected individuals.

Because P6C inactivates PLP and causes cellular depletion of this enzymatic cofactor, a number of biochemical abnormalities occur that are associated with secondary deficiencies of PLP-dependent enzymes, mainly affecting amino acid metabolism. **Table 2** lists some amino acid changes reported in PDE-ALDH7A1 patients and possible links to PLP-dependent enzymes in their metabolic pathways.


*\*Amino acid changes were retrieved from the case series of Mills et al. [75] and Yuzyuk et al. [85].*

↑*: elevated,* ↓*: lowered.*

*\*\*Unless another source is specified, information on PLP-dependent enzymes and their catalytic activities were collectively retrieved from the review of Wilson et al. [6] and KEGG pathway database [88].*

#### **Table 2.**

*Amino acid changes in PDE-ALDH7A1 and related PLP-dependent enzymes.*

#### *2.1.4 Treatment and its outcome*

In patients with PDE-ALDH7A1, seizures are effectively controlled by PN treatment in about 90% of cases [6]. Patients require life-long intake of pharmacological doses of PN for seizure control as PN withdrawal leads to seizure recurrence [60]. In a subset of patients with ATQ deficiency, better seizure control is achieved when folinic acid is added to the PN regimen (known as folinic acid-responsive seizures or FARS) [60]. The subset of FARS patients can be distinguished by the appearance of a characteristic peak (Peak X) on CSF biogenic amine neurotransmitter analysis [60, 89].

Despite effective control of seizures with PN, treatment outcome is usually still poor, and a large proportion of children with PDE-ALDH7A1 have neurodevelopmental impairments [77]. It has been suggested that PN treatment alone cannot prevent the accumulation of high levels of lysine metabolites (P6C, α-AASA and PIP) in the brain which may have neurotoxic effects [90].

To limit the accumulation of these metabolites, substrate (lysine) reduction therapies have been implemented. These consisted of lysine-restricted diet [91], arginine supplementation [92] and triple therapy [93]. Arginine is a natural antagonist of lysine because the two amino acids use the same transporter (known as the y + system) for their transportation across the BBB. Therefore, it was suggested that arginine could compete with lysine and limit its entry to the brain [72, 92]. Triple

*Vitamin B6 and Related Inborn Errors of Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99751*

therapy refers to a combination therapy of lysine-restriction and arginine supplementation (in addition to PN treatment, therefore it was termed "triple therapy") [93]. Clinical trials using these dietary therapies reported reduction in lysine metabolite levels and improvements in the neurodevelopmental outcome in most treated patients [79, 85, 91, 93–96].

### **2.2 PLP-dependent epilepsy (PNPO deficiency)**

### *2.2.1 Disease mechanism*

PNPO catalyzes the rate-limiting step in the biosynthetic pathway of PLP from other vitB6 vitamers (salvage pathway, **Figure 2**). Patients affected with pathogenic variants in its encoding gene, *PNPO*, have reduced activity of PNPO which leads to dysfunction of the salvage pathway and inability of the patients to produce adequate amounts of PLP [86].

#### *2.2.2 Clinical features*

Similar to PDE-ALDH7A1, PNPO deficiency is characterized by early onset, drug-resistant epileptic encephalopathy [87]. Since the disease gene discovery in 2005 [57], about 90 cases of PNPO deficiency have been reported in the medical literature with a phenotypic spectrum that extends from early postnatal lethality to milder forms with well-controlled seizures and normal neurodevelopmental outcome [88, 97–99]. Prematurity is observed in about 50% of the PNPO deficiency cases [88]. Seizures usually start very early after birth (within the first day of life in about 60% of the cases), but can also have a later onset within the first 6 months of life [86, 88]. *In utero* onset of seizures have been suspected in some of the documented cases [87]. PNPO-deficient patients may also suffer from variable degrees of morphological brain defects, most commonly diffuse brain atrophy, and neurodevelopmental deficits [88] as well as systemic co-morbidities such as lactic acidosis, hypoglycaemia, coagulopathy, anemia and ocular and cardiac problems [6, 37, 86, 98].

#### *2.2.3 Biochemical features and diagnostic biomarkers*

PNPO deficiency is associated with a number of biochemical alterations most commonly affecting biogenic amine neurotransmitters. The PLP-dependent enzyme, AADC, plays a central role in the biosynthetic pathway of these neurotransmitters (**Figure 5**). A number of amine neurotransmitter metabolites in this pathway were found to be present at abnormal levels in PNPO-deficient patients, suggesting an impaired flux through the AADC catalyzed step. For example, elevated levels of 3-*O*-methyldopa (3-OMD) and vanillactic acid (VLA) have been frequently detected in patients' CSF and urine samples, respectively [6, 88]. Both compounds are metabolites of L-dopa, the direct precursor of dopamine, which are generated upstream of AADC [44] (**Figure 5**). On the other hand, low CSF concentrations have been detected for metabolites downstream to AADC, namely, homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) [88, 100], the catabolic products of dopamine and serotonin, respectively (**Figure 5**) [44].

The biochemical spectrum of PNPO deficiency also includes amino acid and vitB6 vitamer perturbations. Elevated concentrations of threonine, glycine, histidine and taurine and low concentrations of arginine in CSF and/or plasma

have been all reported in patients [6, 10, 88]. Unlike PDE-ALDH7A1, systemic PLP deficiency is a typical finding in PNPO deficiency as evidenced by the detection of low PLP levels in pre-treatment patient samples (CSF and/or plasma) [37, 88, 101]. Another common vitamer finding is the accumulation of PM, the precursor of PNPO substrate, detected in both pre- and post-treatment plasma samples [101, 102].

Currently there is no specific diagnostic biomarker for PNPO deficiency and genetic testing of the *PNPO* gene is required to establish diagnosis [86]. Altered biogenic amine profile along with low PLP and/or elevated PM in patient biofluids have been proposed as indicators of PNPO deficiency [6, 10, 88]. In a small cohort of patients, Mathis et al. [101] noted a consistently elevated plasma PM/PA ratio irrespective of vitB6 treatment status. This distinct vitamer profile was only observed in PNPO-deficient patients but not in other vitB6EE forms and was therefore suggested to be a candidate biomarker for PNPO deficiency, but this is yet to be validated in a larger cohort of patients [101]. Recently, a new and rapid mass spectrometry-based method has been developed for diagnosis of PNPO deficiency in dried blood spots which relies on measurement of enzyme activity [103].

#### *2.2.4 Treatment and its outcome*

Seizures are usually controlled by supplementation of pharmacological doses of PLP or PN. Based on early reports [57, 104, 105], PNPO deficiency has for some time been viewed as a disease that is only treatable by PLP but not PN (and hence was given the name "PLP-dependent epilepsy"). This was also consistent with the notion that the defective enzyme, PNPO, in these patients is unable to convert supplemented PN to PLP which explains the lack of response to PN treatment. However, it was later found that a subset of affected children (about 40% of cases [6]) show better clinical response to PN while PLP may in fact exacerbate their seizures [87, 106]. Mills et al. [87] suggested that certain genotypes (namely R225H/C and D33V) seem to be more likely to benefit from PN treatment. This was attributed to possible residual enzyme activity that is associated with these *PNPO* mutations and that PN may also have a chaperone-like stabilizing effect on the mutant protein. PLP, on the other hand, may exert an inhibitory effect on the protein and abolish its presumed residual activity leading to more deleterious consequences [106, 107]. Based on treatment response, PNPO-deficient patients appear to fall into at least 3 groups; patients who respond to PLP but are refractory to PN, patients who respond to both vitamers (PLP and PN) and patients who respond to PN but decline upon switching to PLP [86].

In some patients, better seizure control was achieved by adjunct treatments like anti-seizure drugs [106] and/or riboflavin [108] in combination with vitB6 therapy. Riboflavin is a precursor of flavin mononucleotide (FMN), the cofactor of PNPO, and therefore may enhance residual enzyme activity [87]. There were multiple reports of liver problems in patients receiving PLP treatment, and these were linked to possible toxic effects of chronic PLP administration, an observation that warrants careful mentoring of PLP-treated patients [109–111].

Neurodevelopmental outcome is still poor in a large proportion of affected children. A recent literature survey of 87 cases of PNPO deficiency [88] found that 56% of patients suffered developmental and/or intellectual deficits in spite of adequate seizure control with vitB6 therapy. Other reports suggested that early diagnosis and initiation of treatment could lead to normal developmental outcome [4, 109, 112].
