Phenylalanine hydroxylase

Phenylalanine hydroxylase: a biomarker of disease susceptibility in Parkinson’s disease and Amyotrophic lateral sclerosis

Glyn B. Steventona* and Stephen C. Mitchellb

aADMET Solutions Ltd, Ivar Gardens, Basingstoke, Hampshire, RG24 8YD, UK.
bImperial College, Department of Computational and Systems Medicine, Faculty of Medicine, South Kensington, London, SW7 2AZ, UK.

* Corresponding author: [email protected] (GB Steventon)
The S-oxidation of S-carboxymethyl-L-cysteine has been reported previously to be a biomarker of disease susceptibility in Parkinson’s disease and Amyotrophic lateral sclerosis. In this investigation, the original observations have been confirmed with the incidence of the poor metaboliser phenotype (no urinary recovery of S-oxide metabolites) being found to be 3.9% within healthy control population. However, 38.3% of the Parkinson’s disease subjects and 39.0% of the Amyotrophic lateral sclerosis group were phenotyped as poor metabolisers. The consequent odds risk ratio of developing Parkinson’s disease was calculated to be 15.5 (95% CI 9.5-25.3) and for Amyotrophic lateral sclerosis was 15.2 (95% CI 8.8-26.5). Thus, the possible role of the enzyme responsible for the S-oxidation biotransformation reaction, phenylalanine hydroxylase, must be further investigated to elucidate the mechanism(s) of toxicity in susceptible individuals displaying these diseases. A dual role potentially explaining of the role of phenylalanine hydroxylase as a biomarker of disease susceptibility is presented together with the observation that metabolomics is a possible way forward in the identification of potential pro-toxins/toxins in those individuals phenotyped as poor metabolisers (Controls, Parkinson’s disease and Amyotrophic lateral sclerosis subjects).


Parkinson’s disease
Parkinson’s disease (PD), the most common movement disorder and the second most common neurodegenerative disease after Alzheimer’s disease, is characterized primarily by the loss of dopaminergic neurons in the substantia nigra pars compacta leading to a dopamine deficiency in the striatum. The consequential loss of regulation of basal ganglia circuitries accounts for the most prominent motor symptoms, including bradykinesia, hypokinesia, rigidity, resting tremor and postural instability. In addition to the typical motor symptoms, various non-motor features may develop, such as autonomic dysfunction, sleep disturbances, depression and cognitive impairment, indicating a more widespread degenerative process. A pathological hallmark of sporadic PD is the presence of proteinaceous deposits within neuronal perykarya (Lewy bodies) and processes (Lewy neurites), mainly composed of ?-synuclein, ubiquitin, neurofilaments and molecular chaperones 1.
Little is known about the aetiopathogenesis of PD. The most common sporadic form of PD seems to be a complex multifactorial disorder with variable contributions of environmental factors and genetic susceptibility. Aging is the most important risk factor, thus with increasing average life expectancy the incidence and prevalence of PD will rise considerably in the near future. A major breakthrough in PD research was the identification of genes which are responsible for monogenic familial forms. Mutations in the genes encoding ?- synuclein and LRRK2 (leucine-rich repeat kinase 2) are responsible for autosomal dominant forms of PD, presumably by a gain-of-function mechanism. Loss-of-function mutations in the genes encoding parkin, PINK1, and DJ-1 mediate autosomal recessive PD. Sporadic and monogenic forms share important clinical, pathological and biochemical features, notably the progressive demise of dopaminergic neurons in the substantia nigra. Therefore, insight into the function and dysfunction of PD-associated gene products can help to elucidate the underlying mechanisms leading to neuronal cell death. Accumulating evidence indicates that PD-associated genes directly or indirectly impinge on mitochondrial integrity, thereby providing a link to pathophysiological alterations observed in sporadic PD 2-9.

Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving both upper motor neurons (UMN) and lower motor neurons (LMN). UMN signs include hyperreflexia, extensor plantar response, increased muscle tone, and weakness in a topographic representation. LMN signs include weakness, muscle wasting, hyporeflexia, muscle cramps, and fasciculations. Initial presentation varies. Affected individuals typically present with either asymmetric focal weakness of the extremities (stumbling or poor handgrip) or bulbar findings (dysarthria, dysphagia). Other findings may include muscle fasciculations, muscle cramps, and labile affect, but not necessarily mood. Regardless of initial symptoms, atrophy and weakness eventually affect other muscles. Average disease duration is about three years, but it can vary significantly. Death usually results from compromise of the respiratory muscles. The number of individuals newly diagnosed with ALS each year is 1-3:100,000.
The prevalence of individuals with ALS is roughly 4-8:100,000 10 and is similar to the number of newly diagnosed individuals each year because people generally live only two to five years after the diagnosis of ALS is established. The mean age of diagnosis in sporadic ALS is 56 years; individuals age 80 years and older have a standardized incidence of 10.2:100,000 in men and 6.1:100,000 in women 11. Ethnic presentation of ALS worldwide is equal with the exception of the South Pacific (Guam) where the incidence of an ALS/parkinsonism/dementia complex is higher 12, 13. Although recent studies suggest a lower incidence in African populations, these need to be verified by population-based prospective studies.
Like PD little is known about the aetiology of sporadic ALS with reports of environmental or acquired causes of ALS including mercury, manganese and products used in farming (fertilizers, insecticides, herbicides) and dietary factors 14. Genetics also plays some role in the disease pathology with an estimated 10% of individuals with ALS having at least one other affected family member. The enzyme superoxide dismutase (SOD) is particularly associated with ALS1. SOD1 pathogenic variants account for only approximately 20% of all familial ALS and approximately 3% of sporadic ALS 15, 16. Simplex cases, those involving individuals with no family history of ALS with SOD1 pathogenic variants are most likely the result of incomplete penetrance or incomplete family history information, although de novo mutation, observed in one family with the His80Arg variant 17, provides another possible explanation. The age of onset of ALS1 is poorly correlated with genotype and intrafamilial variability can be extensive 18, 19. Thus, the aetiology of sporadic ALS is thought to be multifactorial with a combination of oxidative stress, glutamate excitotoxicity, mitochondrial dysfunction, inflammation, apoptosis and environmental triggers having repeatedly been proposed 20.
Xenobiotic metabolism and degenerative neurological disease
Since 1988 publications have been appearing in the literature indicating that sporadic PD and ALS are associated with abnormalities in function of phenylalanine hydroxylase (PAH) 21-24. No real advances were reported after 2003 and like the ring of power in Tolkein’s Lord of the Rings triology “… some things that should not have been forgotten were lost. History became legend. Legend became myth.” The story of the S-oxidation polymorphism and PD and ALS slowly faded with time. However, following the identification of the enzyme responsible for the sulfur oxygenation of SCMC in rat, mouse, HepG2 cells, humans and cDNA expressed PAH proteins new life has been given to this story 25-34.

It is proposed that the enzyme known as phenylalanine hydroxylase (phenylalanine monooxygenase, PAH, EC. whose classical function is the conversion of phenylalanine to tyrosine, has other metabolic roles within the body. Although phenylalanine is the preferred substrate the enzyme has been shown to oxidise several other compounds including the drug, S-carboxymethyl-L-cysteine. Whilst most allelic variants of PAH adequately metabolise phenylalanine, thereby avoiding any clinical consequence, they are unable to effectively metabolise these secondary substrates. Poor sulphoxidation of S-carboxymethyl-L-cysteine has been reported as a risk factor for developing PD and ALS, the drug being a presumed metabolic ‘probe’ for PAH allelic variants. It is proposed that these allelic variants of PAH, themselves capable of maintaining sufficient phenylalanine metabolism, are unable to metabolise other as yet unknown ‘toxic substances’, and it is the accumulation of subsequent ‘toxic insults’ that assist in the development of these neurological problems.
The S-oxidation polymorphism
A re-evaluation of the S-oxidation polymorphism in controls, PD and ALS subjects has been undertaken to investigate the association of PAH with these diseases. The collated data now re-evaluated has been published previously in various forms 21-24, 35, 36 but never has the data been looked at in its totallity. This resulted in the analysis of 300 healthy controls, 401 disease controls (these included neither degenerative neurological disease subjects nor subjects taking medication). Typical diagnoses in these two groups were lumbar disc disease, benign intracranial hypertension, multiple sclerosis and those in whom no organic disease was discovered. Other in-patients studied had no neurological disease but had diverse general medical illness and were chosen because they were receiving no medication, 175 PD (receiving no medication) and 105 ALS subjects (receiving no medication). The results can be seen in Tables 1, 2 and Figure 1). There were no differences between the healthy controls and disease controls with respect to the statistical analysis of the % total urinary recovered, % sulfides recovered, % S-oxides recovered and S-oxidation Index (Table 1, P;0.05, Kruskal-Wallis One Way Analysis of Variance on Ranks for all variables data not shown). Thus, the results from the heathy controls and the diseased controls was combined to give a control population of 701 subjects. When the statistical analysis of the Controls, PD and ALS subjects was evaluated for male versus female differences with respect to the variables, % total urinary recovered, % sulfides recovered, % S-oxides recovered and S-oxidation Index no significant differences were found (Table 1, P;0.05, Kruskal-Wallis One Way Analysis of Variance on Ranks for all variables data not shown). Thus, the data from male and female subjects within each subject group (Controls, PD and ALS) were combined. The results of the S-oxidation phenotyping study in the Control, PD and ALS subjects can be seen in Tables 1, 2 and Figure 1. There were no significant differences in the total recovery of drug related material (SCMC plus sulfide and S-oxide metabolites) between the Controls (51.3% (17.3%-98.2%)), PD (47.9% (22.9%-94.2%)) and ALS subjects (48. 5% (28.8%-90.8%) P;0.05, Kruskal-Wallis One Way Analysis of Variance on Ranks. However, the % recovery of sulfides was significantly higher in the PD (44.5% (9.3%-90.1%)) and ALS (45.0% (18.9%-90.1%)) subjects compared to the Controls (36.7% (9.3-93.9%)) P