HEALTH SAFETY ASPECTS OF FOODSTUFFS INTENTED FOR PHENYLKETONURICS

Phenylketonuria is a rare metabolic disorder that occurs due to the lack of liver enzyme phenylalanine hydroxylase. Untreated it leads to mental retardation, delayed development of speech, microcephaly, epilepsy, behavioral problems etc. Its treatment consists of strict diet, very low in phenylalanine content. The aim of this study was to determine the health safety and suitability of 17 selected foods labeled as „low in protein“ respectively „foods low in phenylalanine“. Analyses were focused on their phenylalanine content and aminoacid representation. The other aim was to compare phenylalanine measured with the content declared by the producers on their label. By product origin, the largest amount of foodstuffs originated from Poland (59%), followed by Germany (23%) and equal representation (6%) had Slovakia, Hungary and Sweden. Automatic analyzer AAA 400, using ion exchange chromatography, analyzed the samples. The lowest determined phenylalanine content was 1 mg.100g -1 (in rubber candy) and the highest 299 mg.100g -1 (in paté). Powdered egg alternative was monitored as the second highest source of phenylalanine. Phenylalanine content in the monitored foods ranged from 1 to 299.6 mg. 100 g -1 respectively, and increased in the following order: Gum candies (1 mg.100g -1 ) < Cherry jelly (1.4 mg. 100g -1 ) < Flour (5.8 mg.100g -1 ) < Flour (5.9 mg.100g -1 ) < Spaghetti (8.7 mg.100g -1 ) < Pasta (9.9 mg.100g -1 ) < Waffle (14.2 mg.100g -1 ) < Salty sticks (23.8 mg.100g -1 ) < Chocolate (24.1 mg.100g -1 ) < Bread (26 mg.100g -1 ) < Breadcrumbs (27.1 mg.100g -1 ) < Sticks with salt (30.3 mg.100g -1 ) < Ice Cornets (30.8 mg.100g -1 ) < Walnut cookie (37.2 mg.100g -1 ) < Instant soup with noodles (46.1 mg.100g -1 ) < Powdered egg alternative (58 mg.100g -1 ) < Pate (299.6 mg.100g -1 ). Only seven products of 17 observed showed low phenylalanine content, less than 20 mg.100g -1 .


INTRODUCTION
Phenylketonuria (PKU) is an autosomal recessive inherited disorder of phenylalanine metabolism due to a lack of the enzyme phenylalanine hydroxylase.Most forms of PKU and hyperphenylalaninemia (HPA) are caused by mutations in a gene on chromosome 12 of phenylalanine hydroxylase (PAH) (Burnett et al., 2008).Currently, more than 400 known mutations are identified in the gene for PAH.Lack of or insufficient activity of the phenylalanine hydroxylase causes accumulation of phenylalanine (Phe), and phenyl ketone excretion in the urine.Any increase in the concentration of phenylalanine in the blood results in central nervous system damage (Strnová and Ürge, 2007).
Phenylalanine (Phe) is an essential amino acid that is a precursor of melanin, dopamine, and thyroxine.It is an essential amino acid and its natural occurrence in food is usually sufficient (mean content 3.5%).In protein foods, Phe constitutes about 4-5% (Velíšek and Hajšlová, 2009).The artificial sweetener aspartame is also high source of phenylalanine.The ADI for aspartame for healthy person is 40 mg/kg body weight (EFSA, 2014).
In PKU patients, a restriction of foods rich in protein (meat, fish, eggs, bread, dairy products, nuts and seeds), as well as avoidance of drinks containing aspartame help to control blood phenylalanine levels.The developing fetus of women suffering from PKU is particularly sensitive to their mother's phenylalanine levels (EFSA, 2014).The basic treatments of PKU are mixture preparations of aminoacids.Their use ensures an adequate supply of amino acids without phenylalanine, thereby compensating reduced protein intake in low protein dietary regime.Patients with phenylketonuria usually get these foods by medical prescription as most of these foodstuffs can belong to the group of foods for special medical purposes (Strnová and Ürge, 2007), which safety is defined by the Commission Directive 1999/21/EC.Untreated children with persistent hyperphenylalaninemia (PKU) show brain damage.Symptoms include microcephaly, epilepsy, mental disability and behavioral problems.By the use of neonatal screening and early dietary intervention, children born with PKU can live a relatively normal life (Burnett et al., 2008;Mitchell, 2000).
The application of diet at an early age of life can significantly reduce mental deficiencies associated with phenylketonuria.Dietary measures are based on the foods containing a minimum of Phe, while preserving the normal content of tyrosine and other proteins in food.This is often achieved by substitution of phenylalanine free proteins.The aim of this work was to analyze selected amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine) in seventeen food samples, declared by the producer as "Low-protein foods" respectively "Foods low in phenylalanine content " by automatic analyzer AAA 400 and consequently to evaluate their safety and suitability for a particular nutritional use.To compare the content of phenylalanine declared on the label with content of phenylalanine measured.

MATERIAL AND METHODOLOGY
As material used were the foodstuffs offered for phenylketonurics obtained from internet sale and labeled as food suitable for phenylketonurics as "low in protein" respectively "foods low in phenylalanine".Overview of the material used is given in Table 1.By origin of products, most of them originated from Poland (59%), followed by Germany (23%) and equal representation (6%) had Slovakia, Hungary and Sweden.

Statistical analysis of results
The results were evaluated by calculating the mean, coefficient of variation and standard deviation using the statistical software Statistica 8.0.

RESULTS AND DISCUSSION
Treatment of phenylketonuria should be a combination of several diets that reduce the amount of phenylalanine: gluten-free diet, lactose-free and low proteins diet which is the most important.The principle of the diet is to reduce the amount of phenylalanine in the body and to create a balance between its income and the potential use (Komárková and Pazdírková, 2010; Dvořák, 2009).

16.
17. Contrary, as sample with the highest content of phenylalanine (299.6 mg.100g -1 ) was determined pate produced in Germany.The highest amino acid content was also detected (6238.90 mg.100g -1 ) in this sample probabaly due to meat present, and the highest content of glutamic acid (916.62 mg.100g -1 ) among amino acids was determined as well.

Content
Powdered egg alternative (sample no.7) was monitored as the second highest source of phenylalanine.It consists of cornstarch, mono and diglycerides of fatty acids, palm oil, xanthan, rice flour and pea protein.Glutamic acid also prevailed in this sample (Table 3).
The only product of Slovak origin was the sample no. 3, spaghetti.Total sum of amino acids 127.91 mg.100g -1 was observed, which can be assumed together with the observed phenylalanine content (8.75 mg.100g -1 ) as low content and therefore this product can be considered as suitable for consumption by phenylketonurics.
As the most suitable for fenylketonurics with the lowest content of phenylalanine were observed gum candies and cherry jelly (less than 2 mg.100g -1 ).Total aminoacid content detected in these two products was also the lowest, at gum candies 12.04 mg.100g-1 and cherry jelly 14.81 mg.100g -1 .Sample no. 8 was the sticks of Polish origin.They are comparable with the sample no. 5, which are also sticks with salt with a similar composition but made from other producer.Phenylalanine content determined was 30.0 resp.23.8 mg.100g -1 .
As it was stated previously, foods for phenylketonurics are often included in the category of foods for special medical purposes that means a category of foods for particular nutritional uses specially processed or formulated and intended for the dietary management of patients and to be used under medical supervision.They are intended for the exclusive or partial feeding of patients with a limited, impaired or disturbed capacity to take, digest, absorb, metabolise or excrete ordinary foodstuffs or certain nutrients contained therein or metabolites, or with other medically-determined nutrient requirements, whose dietary management cannot be achieved only by modification of the normal diet, by other foods for particular nutritional uses, or by a combination of the two (Commission Directive 1999/21/EC).The labelling of foodstuffs intended for particular nutritional uses shall include by the Directive 2009/39/EC the particular elements of the qualitative and quantitative composition or the special manufacturing process which gives the product its particular nutritional characteristics.Comparing the results in phenylalanine content measured and the content of phenylalanine labeled by producers, we observed several differences shown in the Fig. 1.Sample no. 2   9. 10. 11. 12. 13. 14. 15. 16. 17.

CONCLUSION
In this work, amino acids representation, focused on phenylalanine content in selected samples intented for phenylketonurics was observed.We can conclude that may be due to the lack of legislation requirements, most of observed foods was high in content of phenylalanine.Only seven products of 17 observed showed lower phenylalanine content, less than 20 mg.100g -1 .The obtained results also suggest that certain foods at the content of phenylalanine differ from those listed on the product label.

Table 1
Overview of the material tested..dm - ) and 115 ±2 °C.After the hydrolysis, the test tubes were cooled down to 20°C.Hydrochloric acid was evaporated and the ropy residue was diluted in loading buffer in a 25 cm -3 volumetric flask.The mixture was filtered through 0.22 m filter and loaded into an analyser.