A PUBLIC HEALTH PERSPECTIVE ON THE IMPORTANCE OF PLASMA PHENYLALANINE AND TYROSINE DETERMINATION IN RELATION TO NEWBORN SCREENING AND MONITORING OF TREATMENT IN PHENYLKETONURIA

Abstract

Newborn screening of phenylketonuria (PKU) is performed in many countries, including Romania, in addition to screening for congenital hypothyroidism. Patients affected by PKU require frequent measurements of phenylalanine (Phe) level in blood plasma. Such a determination is important not only in early diagnostic, but also in monitoring the treatment of PKU to maintain phenylalaninemia within limits that will not affect the brain. A simple, highly sensitive, accurate and rather inexpensive procedure for the simultaneous determination of Phe and Tyr plasma concentrations was previously described in this journal. The new procedure may be applied in many clinical laboratories, including those with no previous experience in diagnosis of inherited amino acid metabolic disorders. In this way the major public health problems linked to PKU not being detected in the first weeks of life (including the burden of institutionalized children with preventable mental retardation) may be avoided.

INTRODUCTION

Screening was defined as the systematic examination or testing of a group of individuals to identify those who either have an undiagnosed disease or defect, or are at sufficient risk of a specific disorder in order to benefit from further investigation or direct preventive action (1). Newborn screening (NBS) is a public health program designed to screen infants shortly after birth for developmental, genetic, and metabolic disorders that are treatable, but not clinically evident in the newborn period (2, 3). NBS is of great importance because an early risk identification allows steps to be taken before symptoms develop (2 - 4). Although each of these illnesses is rare, taken together they represent a public health problem; consequently, NBS saves lives or improves quality of life (1). Screening programs are usually run by national governing bodies with the goal of screening all infants born in the jurisdiction and the number of diseases screened can vary greatly, mainly due to financial reasons (2 - 4).

Newborn screening as a public health program

Newborn screening debuted as a public health program in the United States in the early 1960s, and has expanded to countries around the world, with different numbers of diseases tested in each country; in most countries NBS includes testing for phenylketonuria (PKU) and congenital hypothyroidism (2 - 4). NBS has evolved from a simple blood or urine screening test to a more comprehensive and complex screening system capable of detecting over 50 different conditions (5). However, this requires very well equiped laboratories and expert personnel, making NBS very expensive. Implementation of NBS to developing countries worldwide should be considered as a priority from the public health perspective (2 - 4).

Groselj et al. (6) assessed the current state of NBS in the region of southeastern Europe, as an example of a developing region. Responses were obtained from 11 countries. PKU screening was not introduced in four of 11 countries, while congenital hypothyroidism screening was not introduced in three of them.

As explained by Public Health England (4) the screening process can be compared to putting people through a sieve, representing the test and most people pass through it (i.e. they are at low risk of having the condition screened for). The people left in the sieve are at higher risk of having the condition. A further investigation is then offered to them. Identification through this process can show that they have the condition screened for. The person may need further confirmatory diagnostic tests. Follow-up testing is typically coordinated between geneticists and the infant’s pediatrician.

Screening for phenylketonuria

The first disorder detected by modern newborn screening programs was PKU, the most common inborn error of amino acid metabolism in which the inability to degrade the essential amino acid phenylalanine (Phe) is caused by absent or virtually absent phenylalanine hydroxylase (PAH) enzyme activity, which normally converts Phe to tyrosine (Tyr). This leads to abnormally high levels of Phe to accumulate in the blood, which is toxic to the brain (7), causing irreversible mental retardation unless detected early. Fortunately, PKU may be diagnosed at birth and may be treated by dietary means (8). If the treatment is started in the first weeks of life and is well monitored the cerebral damage can be largely eliminated.

Robert Guthrie developed a simple method using a bacterial inhibition assay that could detect high levels of phenylalanine in blood shortly after a baby was born. Guthrie also pioneered the collection of blood on filter paper which could be easily transported, recognizing the need for a simple system if the screening was going to be done on a large scale. Newborn screening around the world is still done using similar filter paper (3, 4).

Romania started the national program for screening of congenital hypothyrodism and PKU in 1999. Phe and thyroid stimulating hormone (TSH) are tested by fluorometric assay. Babies with hypothyroidism do not make enough thyroid hormone and without treatment can have slowed growth and brain damage. Taking thyroid hormone medication shortly after birth can prevent these problems (2).

Patients affected by PKU require frequent measurements of Phe level in the blood plasma. Such a determination is important not only in early diagnostic, but also in monitoring the treatment of PKU, since maintaining the plasma Phe level within certain ranges (between 2 and 6 mg/dL, or equivalently 120 and 360 µmol/L ) is the key point during treatment for clinicians to achieve the best long-term neuropsychological outcome (7, 8).

A simple, highly sensitive and accurate procedure for the simultaneous determination of Phe and Tyr plasma concentrations was previously described (9, 10). It involves two steps: a) separation of plasma, isolation and preparation of a concentrated solution of amino acids (by ion-exchange column chromatography, evaporation of the eluate in vacuum at 40 ¯C, and solubilization of the residue in double distilled water), performed essentially as first described by Wadman and coworkers (11, 12), and b) determination of Phe and Tyr concentrations by HPLC.

The new procedure (9, 10) is rather inexpensive and may be applied in many clinical laboratories, including those with no previous experience in diagnosis of inherited amino acid metabolic disorders. Groselj et al. (13) emphasized that after 50 years of PKU newborn screening there are still many countries that cannot afford to benefit of this because of the high cost of modern methodology of detection of hyperphenylalaninemia, followed by monitoring the Phe concentration in the blood. In case of the methodology previously described (9, 10) the isolation of amino acids from plasma (the step a) may be performed by personnel with various backgrounds working in clinical laboratories (biologists, chemists, technicians) after short time training. The determination of Phe concentration in blood by HPLC (step b) requires trained personnel and more expensive equipment compared with the column chromatography. However, the procedure described here does not require special columns for amino acid analysis, so that any laboratory having a HPLC instrument may set up the procedure. In addition, the determination of plasma Phe by HPLC as presented here has the advantage of being fast, very sensitive and highly accurate.

In conclusion, the methodology for determination of plasma Phe and Tyr concentrations previously described (9, 10) is very adequate, not only for confirming the diagnosis of PKU in patients with neonatal hyperphenylalaninemia, but also for monitoring the plasma concentrations of Phe and Tyr in patients with PKU (to maintain phenylalaninemia within limits that will not affect the brain). In this way the major public health problems linked to PKU not being detected in the first weeks of life (including the burden of institutionalized children with preventable mental retardation) may be avoided.

Conflict of interest

The authors declare that they have no conflict of interest concerning this article.

Acknowledgements

The corresponding author is grateful to his former co-workers with whom the methods of analysis of amino acids were first introduced in the medical laboratories in Romania (14, 15). He is also grateful to the late Professor S.K. Wadman (Laboratory of the Wilhelmina Kinderziekenhuis, Utrecht, The Netherlands) and his co-workers for their kindness to teach him the methods for analyses of amino acids for performing the diagnosis of amino acid metabolism disturbances.