Health safety aspects of foodstuffs intented for phenylketonurics

Potravinarstvo® Scientific Journal for Food Industry Volume 9 132 No. 1/2015 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. Dietary restriction of natural proteins reduces and Potravinarstvo, vol. 9, 2015, no. 1, p. 132-137 doi:10.5219/455 Received: 9 March 2015. Accepted: 6 May 2015. Available online: 25 May 2015 at www.potravinarstvo.com © 2015 Potravinarstvo. All rights reserved. ISSN 1337-0960 (online) License: CC BY 3.0 HEALTH SAFETY ASPECTS OF FOODSTUFFS INTENTED FOR PHENYLKETONURICS Stanislava Matejová, Martina Fikselová, František Buňka ABSTRACT 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. Keywords: phenylketonuria; phenylalanine; low-protein product; acid hydrolysis; chromatography Potravinarstvo® Scientific Journal for Food Industry Volume 9 133 No. 1/2015 maintains the level of Phe in the blood (Macdonald et al. 2009; Poustie and Wildgoose, 2010; Svačina, 2008). 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. Fifteen amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, histidine, lysine and arginine) were determined in observed samples using ionexchange chromatography. The total amount of amino Table 1 Overview of the material tested. No. Product Producer Main components 1. Pate Producer A, Germany bacon, pork, liver, onion, water, salt, stabilizer E 250, spice, emulsifier 2. Breadcrumbs Producer 1, Poland gluten free wheat starch, corn starch, vegetable oil, glucose, sugar, yeast, salt, guar gum, E 464, fibre, raising agents, E 500, E 575 3. Spaghetti Slovakia corn starch, modified corn starch, emulsifier E 471, dye curcuma (E 100) 4. Instant soup with noodles Producer 1, Poland maltodextrin, gluten free pasta, corn starch, water, salt, mono and diglycerides of fatty acids, E 464, E 575, betacarotene, salt, monosodium glutamate, vegetable oil, carrot, sugar, onion, aroma, celery, leek, black pepper, parsley stalk, curcuma, citric acid 5. Sticks with salt Producer 1, Poland corn starch, salt, wheat starch gluten free, vegetable oil, sugar, yeast, guar gum, E 464, salt, fibre, E 500, E 503, mono and diglycerides of fatty acids, lecithin, E 575 6. Walnut cookie Producer 2, Poland corn starch, gluten free wheat starch, vegetable oil, sugar, walnuts, peanuts, oil, pectin, guar gum, pectin, glucose, butter essence, mono and diglycerides of fatty acids 7. Powdered egg alternative Producer B, Germany corn starch, mono and diglycerides of fatty acids, palm oil, xanthan, rice flour, pea protein 8. Salty sticks Producer 2, Poland margarine, corn starch, gluten free wheat starch, water, sugar, potato starch, yolk, guar gum, yeast, potato syrup, wheat gluten free cellulose, salt, E 503, mono and diglycerides of fatty acids, lecithin, vanillin 9. Chocolate Producer C, Germany cocoa butter, sugar, butter, cocoa matter, powdered whey, vanilla 10. Pasta Hungary corn starch, water, fatty acids, E 471 11. Waffle Producer 2, Poland gluten free wheat starch, corn starch, guar gum, bamboo fibre, glucose, apple pectin, mono and diglycerides of fatty acids 12. Cherry jelly Producer 2, Poland sugar, glucose syrup, water, agar, citric acid, cherry aroma, dye E 124 13. Ice Cornets Producer 2, Poland corn starch, gluten free wheat starch, potato starch, sugar, yolk, guar gum 14. Bread Producer 1, Poland gluten free wheat starch, water, vegetable fat, sugar, glucose, yeast, salt, carob, guar gum, E 464, fibre, E 500, E 575 15. Gum candies Producer C, Germany potato and tapioca starch, citric acid, malic acid, galactic acid, calcium citrate, arome, fruit concentrates, vegetable extracts (blackberries, carrot, pepper, curcuma, spirulina, nettle, spinach), natural dye : chlorophyll, vegetable oil, beeswax, carnauba wax 16. Flour Producer 1, Poland gluten free wheat starch, glucose, corn starch, guar gum, E 464, vegetable fibre, E 500, E 575 17. Flour Sweden wheat starch (max. 0.2 g gluten/100 g), oligofructose, guar gum, vitamins, ferrum Potravinarstvo® Scientific Journal for Food Industry Volume 9 134 No. 1/2015 acids and phenylalanine content of food products were monitored after 23-hour acid hydrolysis using HCl (6 mol.dm-3) 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. Amino Acid Analyser AAA400 (Ingos, Prague, Czech Republic) was equipped with a column (370×3.7 mm filled with a ion exchanger Ostion LG ANG – Ingos, Prague, Czech Republic), post-column ninhydrine derivatization and spectrophotometric detection (440 nm for proline and 570 nm for other amino acids). Method was performed according to the Buňka et al. (2009). 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). Phenylalanine content in the monitored foods in our work was represented in varying amounts, its content 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 no.16 (5.8 mg.100g-1) < Flour no. 17 (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). Following legislation, it seems that national or European legislation contain no specific requirements for „low protein foods“, or foods low in phenylalanine, so in purpose to evaluate our samples we used as the standard given in the Czech legislation (Vyhláška Ministerstva zdravotnictví České republiky č. 54/2004 Sb), according to which food with no phenylalanine content is defined as food made by special technological process so that the phenylalanine content should not exceed 20 mg per 100 g or 100 cm-3 in the food intended for consumption. Under this legislation, suitable foods for phenylketonurics could be classified as follows: Gum candies (1 mg.100g-1) < Cherry jelly (1.4 mg. 100g-1) < Flour no. 16 (5.8 mg.100g-1) < Flour no.17 (5.9 mg.100g-1) < Spaghetti (8.7 mg.100g-1) < Pasta (9.9 mg.100g-1) < Waffle (14.2 mg.100g-1). Table 2 Aminoacid content (mg.100g-1) determined in sample of pate. Aminoacids Content of aminoacids [mg.100g-1] Standard deviation CV (%) Aspartic acid 571.11 0.14 2 Threonine 201.60 0.04 2 Serine 156.48 0.04 3 Glutamic acid 916.62 0.55 6 Proline 546.50 0.27 5 Glycine 762.99 0.12 2 Alanine 471.35 0.02 0 Valine 371.65 0.33 9 Isoleucine 269.77 0.15 5 Leucine 522.17 0.19 4 Tyrosine 121.15 0.03 3 Phenylalanine 299.59 0.13 4 Histidine 205.64 0.03 2 Lysine 435.07 0.01 0 Arginine 387.21 0.24 6 Total 6238.90 Potravinarstvo® Scientific Journal for Food Industry Volume 9 135 No. 1/2015 Table 3 Aminoacid content (mg.100g-1) determined in sample of powdered egg alternative. Aminoacids Content of aminoacids [mg.100g-1] Standard deviation CV (%) Aspartic acid 125.69 0.05 4 Threonine 33.60 0.02 7 Serín 55.26 0.03 6 Glutamic acid 163.12 0.08 5 Proline 39.72 0.01 2 Glycine 46.35 0.02 5 Alanine 58.73 0.02 3 Valine 38.43 0.00 0 Isoleucine 29.31 0.02 7 Leucine 87.81 0.04 5 Tyrosine 28.76 0.01 5 Phenylalanine 58.00 0.01 1 Histidine 32.71 0.01 4 Lysine 60.59 0.01 1 Arginine 87.63 0.02 3 Total 945.70 Figure 1 Differences between phenylalanine content measured and labeled by producers (mg.100g-1). Samples: 1.Pate, 2. Breadcrumbs, 3. Spaghetti, 4. Instant soup with noodles, 5. Sticks with salt, 6. Walnut cookie, 7. Powdered egg alternative, 8. Salty sticks, 9. Chocolate, 10. Pasta, 11. Waffle, 12. Cherry jelly, 13. Ice Cornets, 14. Bread, 15. Gum candies, 16. Flour, 17. Flour. 025 50 75 100 125 150 175 200 225 250 275 300 325 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Content of phenylalanine (mg.100g-1) Sample PHE declared PHE measured Potravinarstvo® Scientific Journal for Food Industry Volume 9 136 No. 1/2015 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. 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. Comparable could be also two samples of flours originating from Poland and Sweden, with detected phenylalanine content at flour no.16 (5.8 mg.100g-1), and flour no.17 (5.9 mg.100g-1). Total aminoacid content were observed as similar (85.08 mg.100g-1 vs. 86.65 mg.100g-1) as well. 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 Figure 2 Total aminoacid content (mg.100g-1) determined in selected products. Samples: 1.Pate, 2. Breadcrumbs, 3. Spaghetti, 4. Instant soup with noodles, 5. Sticks with salt, 6. Walnut cookie, 7. Powdered egg alternative, 8. Salty sticks, 9. Chocolate, 10. Pasta, 11. Waffle, 12. Cherry jelly, 13. Ice Cornets, 14. Bread, 15. Gum candies, 16. Flour, 17. Flour. 6238.9 443.13 127.91 2907.39 541.36 678.89 945.7 376.39 385.36 142.54 270.29 14.81 543.08 452.62 12.04 86.65 85.08 0 1000 2000 3000 4000 5000 6000 7000 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Sample Total aminoacid content (mg.100g-1) Potravinarstvo® Scientific Journal for Food Industry Volume 9 137 No. 1/2015 represented, as breadcrumbs and sample no. 6 were the products with the lowest difference in observed and declared values of phenylalanine in the packaging. Contrary, higher differences in these two amounts were detected in samples 1, 4, 7, 8. Total aminoacid content determined in selected products is shown in the Figure 2. Its content in selected products ranged from 12.04 (gum candies) to 6238.90 mg.100g-1 (in pate) and increased in the order: Gum candies (12.04 mg.100g-1) < Cherry jelly (14.81 mg.100g-1) < Flour (85.08 mg.100g-1) < Flour (86.65 mg.100g-1) < Spaghetti (127. 9 mg.100g-1) < Pasta (142.54 mg.100g-1) < Waffle (270.29 mg.100g-1) < Salty sticks (376.39 mg.100g-1) < Chocolate (385.36 mg.100g-1) < Breadcrumbs (443.13 mg.100g-1) < Bread (452.62 mg.100g-1) < Sticks with salt (541.36 mg.100g-1) < Ice Cornets (543.08 mg.100g-1) < Walnut cookie (678.89 mg.100g-1) < Powdered egg alternative (945.70 mg.100g-1) < Instant soup with noodles (2907.39 mg.100g-1) < Pate (6238.90 mg.100g-1). 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. REFERENCES Directive 2009/39/EC of the European Parlament and of the Council of 6 May 2009 on foodstuffs intended for particular nutritional uses, OJ L 124, 20.5.2009, p. 21-29. Buňka, F., Kříž, O., Veličková, A., Buňková, L., Kráčmar, S. 2009. Effect of acid hydrolysis time on amino acid determination in casein and processed cheeses with different fat content. Journal of Food Composition and Analysis, vol. 22. no 3. p. 224-232. http://dx.doi.org/10.1016/j.jfca.2008.10.023 Burnett, J. R., Mamotte, C. D. S., Williams, R. A. 2008. Phenylketonuria: An Inborn Error of Phenylalanine Metabolism. Clin. Biochem. Review. vol. 29, no.1, p. 31-41. 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Cochrane Database of Systematic Reviews. no 1. http://dx.doi.org/10.1002/14651858.CD001304.pub2 PMid:20091517 Strnová, J., Ürge, O. 2007. Maternálna fenylketonúria. (Maternal Phenylketonuria). In Via pract., vol. 4, no. 9, p. 417-419. Svačina, Š., Bretšnajdrová, A., Holcátová, I. et al. 2008. Klinická dietologie (Clinical Dietetics). 1st ed. Praha: Grada Publishing a.s., 384 p. ISBN 978-80-247-2256-6. Velíšek, J., Hajšlová, J. 2009. Chemie potravin. II. (Food Chemistry. II.) 3rd ed Havličkuv Brod: OSSIS. 644 p. ISBN 978-80-86659-16-9. Decree of the Czech Ministry of Health on 30 January 2004 no. 54/2004 Coll. on foodstuffs intended for particular nutritional uses and how they are used. Acknowledgment: This study was supported by a project of the internal grants of Tomas Bata University in Zlin, Czech Republic no IGA/FT/2015/004 funded from the resources for specific university research. Contact address: Stanislava Matejová, Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Hygiene and Food Safety, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, E-mail: s.[email protected]m. Martina Fikselová, Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Hygiene and Food Safety, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, E-mail: [email protected]. František Buňka, Tomas Bata University in Zlín, Faculty of Technology, Department of Food Technology, nám. T. G. Masaryka 5555, 760 01 Zlín Czech Republic E-mail: b[email protected].