Acta Biochimica Polonica, Vol. 65, No 2/2018, 303–308, https://doi.org/10.18388/abp.2018_2565

Regular paper

Deficiency of long-chain polyunsaturated fatty acids in phenylketonuria: a cross-sectional study

Sławomira Drzymała-Czyż1*, Łukasz Kałużny1, Patrycja Krzyżanowska-Jankowska1, Dariusz Walkowiak2, Renata Mozrzymas3 and Jarosław Walkowiak1

1Department of Pediatric Gastroenterology and Metabolic Diseases, Poznan University of Medical Sciences, Poznań, Poland; 2Department of Organization and Management in Health Care, Poznan University of Medical Sciences, Poznań, Poland; 3Voivodeship Hospital, Wrocław, Poland

The etiology of altered blood fatty acid (FA) profile in phenylketonuria (PKU) is understood only partially. We aimed to determine whether FAs deficiency is dependent on the diet or metabolic disturbances. The study comprised 40 PKU patients (20 female, 20 male; aged 11 to 35 years; 12 children and 28 adults) and 40 healthy subjects (HS; 20 female, 20 male, aged 18 to 33 years). We assessed the profile of FAs (gas chromatography/mass spectrometry) and analyzed the 72-hour dietary recalls. The amount of C14:0, C16:0 and C16:1n-7, C18:1n-9 did not differ between the analyzed groups. The percentage of C18:0 was higher, while C20:3n-9, C18:2n-6, C20:2n-6, C20:4n-6, C22:4n-6, C22:5n-6 and C22:6n-3 was lower in PKU than in HS. However, C18:3n-6, C18:3n-3 and n-6/n-3 ratio were higher in PKU patients. The C20:4n-6/C20:3n-6 ratio (reaction catalyzed by Δ5-desaturase), the C22:5n-6/C22:4n-6 and the C22:6n-3/C22:5n-3 ratio (both reactions catalyzed by Δ6 desaturase) were significantly lower in PKU patients. Therefore, the deficiency of long-chain polyunsaturated fatty acids in PKU patients may result not only from inadequate supply but also from metabolic disturbances.

Key words: phospholipids, docosahexaenoic acid, dietary intake, inborn error of metabolism, metabolic diseases, desaturase

Received: 02 February, 2018; revised: 04 May, 2018; accepted: 07 May, 2018; available on-line: 18 June, 2018

*e-mail: slawomiradrzymala@wp.pl

Abbreviations: AA, arachidonic acid; ALA, α-linolenic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; EER, estimated energy requirement; FA, fatty acid; HS, healthy subjects; LA, linoleic acid; LCPUFA, long-chain polyunsaturated fatty acids; MUFA, monounsaturated fatty acids; PKU, Phenylketonuria; PAH, phenylalanine hydroxylase; Phe, phenylalanine; ROS, Rostock-Oseretzky Scale; SFA, saturated fatty acids; VEP, visually evoked potentials

Introduction

Phenylketonuria (PKU) is the most frequent inborn error of amino acid metabolism (Blau et al., 2011; Singh et al., 2016). A deficiency in phenylalanine hydroxylase (PAH) activity leads to elevated levels of phenylalanine (Phe) in the blood. The signs and symptoms of untreated PKU comprise severe encephalopathy, mental retardation and neurological abnormalities, including seizures, microcephaly and other symptoms. Diagnosis by newborn screening and the implementation of a low-Phe diet in the neonatal period and beyond result in normal neurological and intellectual development in PKU patients (Blau et al., 2011; Scriver et al., 2001; Singh et al., 2016). In the low-Phe diet, 60–80% of the daily protein intake derives from a Phe-free l-amino acids mixture and 20–40% from fruit, vegetables and specially manufactured low-protein products (Blau et al., 2010). The parents of PKU patients treated in our metabolic outpatient clinic analyzed the Phe, protein and energy intake of their children (as in most other European countries).

The PKU diet excludes meat and fish, which are natural sources of long-chain polyunsaturated fatty acids (PUFA) (Al Hafid & Christodoulou, 2015; Singh et al., 2016). PUFA deficiency may negatively impact patients’ psychomotor development and may induce metabolic imbalance, thus leading to changes in the fatty acids’ (FA) profile (Al Hafid & Christodoulou, 2015; Gramer et al., 2016; Schindeler et al., 2007). It should be noted that information on the relationship between the low-Phe diet and PUFA profile in PKU is contradictory (Acosta et al., 2001; Giovannini et al., 2011; Htun et al., 2015; Lage et al., 2010; van Gool CJ et al., 2000).

The aim of the study was to verify the hypothesis that the deficiency of specific FAs in PKU is dependent on either metabolic disturbances or the diet. This goal was achieved through the analysis of a larger number of FAs than in most studies which allowed us to investigate the desaturase activity in the context of dietary intakes.

Materials and methods

Patients. The study comprised 40 PKU patients (20 female, 20 male; aged 11 to 35 years; 12 children and 28 adults). The inclusion criteria were: PKU diagnosed by newborn screening and confirmed by biochemical testing, under regular control in an outpatient clinic, the reliable use of a low-Phe diet (good compliance), good general status and a willingness to participate in the study. The exclusion criteria were: elevated Phe levels during the previous 3 months (defined according to the recommendations – Phe concentration in the serum above 600 µmol/L; van Wegberg et al., 2017); the presence of comorbidities (e.g., chronic gastrointestinal diseases or inflammatory processes, such as tonsillitis) and a family history positive for dyslipidaemia (hypercholesterolemia, hypertriglyceridemia), taking supplements enriched with FAs (e.g., blubber).

The control group consisted of 40 healthy subjects (HS; 20 female, 20 male, aged 18 to 33 years). Patients and HS were involved in the study in the years 2014–2015. All analyzes were performed in 2015/2016.

The anthropometric parameters describing PKU patients (n=40) and HS (n=40) are presented in Table 1.

Dietary intake. To estimate energy and macronutrient intake, subjects were asked to complete a 3-day diary (two working days and one during the weekend). The average content of energy, protein (Phe), lipids and carbohydrates in the daily food intake was calculated. The percentage of estimated daily energy requirements (EER) was calculated using Polish references for human nutrition (Jarosz, 2012). The analyses were performed with the use of Dietetyk 2015 software (Jumar, Poznań, Poland).

Additionally, the composition of all available PKU formulas was taken into consideration in order to determine FAs content (the results are presented in Supplementary Table A at www.actabp.pl).

Fatty acid analysis. Blood samples were collected after an overnight fasting period. Samples were centrifuged (3 000 × g, 10 min, 4°C), and stored in plastic vials at –80°C.

The profiles of the FAs were assessed using Glaser’s method as we described previously (Drzymała-Czyż et al., 2017; Glaser et al., 2010). Individual fatty acid methyl esters were quantified by gas chromatography with mass spectrum (Agilent 7890 series II and 5975C, Agilent Technologies, Santa Clara, USA) using a BPX 70 column (BPX70, 25 m × 0.22 mm ID × 0.25 μm, SGE Analytical Science, Ringwood, Australia) as described previously (Glaser et al., 2010). Peak integration was performed using MSD ChemStation (Agilent Technologies, Santa Clara, USA).

Statistical methods. The results of the analysis of the FAs were expressed as percentage values (% wt/wt). For all parameters, the medians and 1st–3rd quartiles were calculated. The Shapiro-Wilk test was used to check the normality of the data distribution. The Mann-Whitney U-test was used to assess the differences between the two groups (Tables 1 and 3). The correlations between daily FAs intake and their levels in serum were analyzed using Pearson’s test. Values of p<0.05 were considered to be statistically significant. All statistical analyses were performed in Statistica 12.0 software environment (StatSoft, Inc., Tulsa, USA).

Ethical considerations. The protocol of the investigation was approved by the Ethical Committee of Poznań University of Medical Sciences, Poznań, Poland (decision no. 383/12). Written informed consent was obtained from the patients’ parents and from patients who were 16-years-old or older. The study was carried out in accordance with the revised Declaration of Helsinki.

Results

The consumption of protein, lipids and carbohydrates was balanced in both groups (Table 2). Daily energy intakes were adequate when compared to EERs.

It should be noted that the consumption of total lipids (including monounsaturated fat and polyunsaturated fat) was lower in PKU patients. The daily intake of C18:2n-6 (linoleic acid; LA) was insufficient in all the patients tested (less than the recommended 4% of total energy), while the supply of C18:3n-3 (α-linolenic acid; ALA) in the diet was sufficient in 61% of patients (more than the recommended 0.5% of total energy) (Jarosz, 2012). Almost half of the daily intake of LA and ALA came from PKU formulas (median [1st–3rd quartile]: 47.4% [32.0–63.3] and 48.1% [28.3–56.9], respectively). The fat content of PKU formulas available in Poland is presented in Supplementary Table A at www.actabp.pl. For comparison, the insufficient consumption of LA and ALA in the control group was noted only in 32% and 3% of subjects, respectively.

The profile of selected FAs in the serum glycerophospholipids of PKU patients and HS is shown in Table 3. The amounts of two saturated FAs (SFA; C14:0, C16:0) and two monounsaturated FAs (MUFA; C16:1n-7, C18:1n-9) did not differ between the groups in the study. The percentage of C18:0 was higher, while C20:3n-9 was lower in the group of PKU patients.

C18:2n-6, C20:2n-6, C20:4n-6, C22:4n-6, C22:5n-6 and C22:6n-3 were lower in the PKU group than in HS. However, C18:3n-6 and C18:3n-3 were higher in PKU patients. The n-6/n-3 ratio was higher in the PKU group.

The C18:3n-6/C18:2n-6 ratio (reaction catalyzed by Δ6-desaturase) was significantly higher in PKU patients compared with HS. However, the C22:5n-6/C22:4n-6 and the C22:6n-3/C22:5n-3 ratio (both also catalyzed by Δ6-desaturase) were significantly lower in PKU patients, similarly as the C20:4n-6/C20:3n-6 ratio (reaction catalyzed by Δ5-desaturase).

No correlations between daily FAs intake and their serum levels were found (ns for all FAs).

Discussion

We performed a comprehensive analysis of serum FA composition including 17 long-chain FAs in PKU patients adhering to the Phe-restricted diet, which revealed abnormalities in long-chain FAs. We documented that changes in the FA profile did not depend on the diet exclusively, but seemed to be also related to metabolic disturbances present in PKU.

The main aim of treating PKU is to keep the serum Phe levels within the recommended range. Brain damage prevention is the main reason for using a protein-, Phe- and energy-balanced diet (Blau et al., 2011; Singh et al., 2016). The PKU-diet is a restrictive diet and, to some extent, resembles a vegan diet. According to the previous studies, vegan-like diets usually tend to be very poor sources of LCPUFAs. They result in poor LCPUFA status and docosahexaenoic acid (DHA) amounts in plasma and erythrocytes (Feillet & Agostoni, 2010; Moseley et al., 2002). This may have negative consequences for the function of the central nervous system, especially in the first year of life. Therefore, it was necessary to implement the supplementation of LCPUFA in infancy by using special Phe-free formulas (Giovannini et al., 2012; Koletzko et al., 2007).

The beneficial effect of PUFA supplementation in PKU was described by Agostoni and coworkers (Agostoni et al., 2003) who found that the latency time in the P100 wave of visually evoked potentials (VEP) improves (decreases) in PKU children who are given eicosapentaenoic acid (EPA) and DHA. However, the effect was not durable and the parameter returned to its baseline value after three years (Agostoni et al., 2003). The authors suggested that the non-persistence of the effect of DHA supplementation could result from the small size of the research population (n=10) and the relatively high intersubject variability. Koletzko and coworkers (Koletzko et al., 2009) analyzed plasma phospholipids FAs, VEP and motor skills by motometric function based on the Rostock-Oseretzky Scale (ROS). VEP and ROS significantly improved after 3 months of DHA supplementation (dose: 15 mg DHA/kg daily), which also resulted in increases in EPA and DHA plasma levels (Beblo et al., 2007; Koletzko et al., 2009). The Committee on Nutrition of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) concluded that LCPUFA supplementation in PKU children older than 2 years, although feasible and safe, offers only transient benefits in the visual function (Agostoni et al., 2011).

The positive effects of PUFA supplementation are well documented. Unfortunately, despite this knowledge, too little attention is paid to it and PUFA deficiency is widespread. Lohner conducted a systematic review and a meta-analysis, which were based on 9 case-control studies and 6 randomized controlled trials, showing a poorer status of both EPA and DHA in PKU patients (Lohner et al., 2013). The failure to obtain optimal PUFA status was explained by the limited dietary intake. PUFA levels depended on the degree of restrictiveness of the diet used, especially in patients older than 2 years. It should be noted that formulas for older children do not provide a sufficient intake of FAs. A low level of arachidonic acid (AA) in total plasma lipids was also described – an observation consistent with our results. Likewise, newer research documented that plasma DHA levels in study subjects were significantly lower than in the control group (Bosdet et al., 2014). On the other hand, higher levels of EPA and ALA in subjects with PKU were found in that study. For this reason, the authors suggested that FAs should be supplemented with preformed DHA rather than with its precursors. In our study, a high percentage of ALA and a low of DHA were also reported.

We did not find any correlation between daily FAs intake and their serum levels. PUFAs intake did not affect their levels in a study by Gramer and coworkers either (Gramer et al., 2016). The authors noted that DHA levels were similar in patients consuming PKU formula with or without PUFA supplementation. This result can suggest that not only dietary but also metabolic disturbances lead to this FA deficiency.

The daily intake of C18:2n-6 (LA) and C18:3n-3 (ALA) should be 4% and 0.5% of the total energy, respectively (Jarosz, 2012). Assuming that the average energy intake was 2000 kcal in our patients, the supply of LA should be 9.0 g and that of ALA 1.1 g. If the patients had been on a PKU-diet and used PKU formulas in a “normal” (standard) dose, they would not consume a sufficient amount of the previously mentioned FAs. Therefore, it seems that the current composition of PKU formulasis insufficient in this respect. Even an extra 10 g of rapeseed oil (most commonly used in the Polish population) does not provide enough LA (such a portion would contain 1.7 g LA and 0.8 g ALA). On the other hand, as we show in Supplementary Table A at www.actabp.pl, the data regarding formula composition are not complete and in many cases, the composition of the formula is not properly defined (different vegetable oils and fats used in different batches). Therefore, it is difficult to reliably assess the total intake of FAs in PKU.

In our study the following ratios were lower in PKU patients than in HS: C20:4n-6/C20:3n-6 (reaction catalyzed by Δ5-desaturase), C22:5n-6/C22:4n-6 and C22:6n-3/C22:5n-3 ratio (both: Δ6-desaturase); on the other hand the C18:3n-6/C18:2n-6 ratio (also Δ6-desaturase) was higher. This might indicate a lack of feedback inhibition of Δ6-desaturase by insufficient DHA (Calder, 2012). Our results were presented as percentages (% wt/wt), which allowed us to highlight the changes in the processing of FAs.

LA and ALA belong to essential fatty acids which cannot be synthesized in the human organism. However, these FAs are the precursors for AA and DHA. DHA is synthesized in two paths – microsomal-peroxisomal and mitochondrial (Infante & Huszagh, 2001; Moore et al., 1995). In different tissue models, Phe and its metabolites inhibited the mitochondrial FA desaturase and the pathway of DHA synthesis (Infante & Huszagh, 2001; Moore et al., 1995). Furthermore, it seems that this process may occur even in patients who use low-Phe diet recommendations (as in our study), because the PUFA levels are substantially lower in PKU patients than those of healthy individuals on vegetarian or similar vegetarian-type diets. Infante and Huszagh (Infante & Huszagh, 2001) suggested that catabolic products of excess Phe (phenylpyruvate and phenylacetate) inhibit alpha-tocopherolquinone synthesis at the level of the deoxygenation reaction which leads to an impairment of DHA and AA synthesis in PKU. Schuck et al. found evidence for the inhibiting effect of Phe metabolites on PUFA synthesis as a potential reason for lipid disturbances (Schuck et al., 2015). In PKU patients total lipid metabolism seems to be impaired, as exemplified by low total cholesterol, high-density lipoprotein, low-density lipoprotein and apolipoprotein A-I/A-II and B. Studies in an animal model showed that these observations may be related to a decrease in expression of an enzyme regulating cholesterogenesis – 3-hydroxy-3-methylglutaryl coenzyme A reductase (Schuck et al., 2015; Shefer et al., 2000). It should be considered that in our study the levels of ALA were higher while DHA was lower and it was accompanied by similar changes in the ratios between the corresponding FAs. This confirms that in PKU patients endogenous synthesis of PUFA can be impaired.

In conclusion, we found that the deficiency of LCPUFAs in PKU patients may result not only from an insufficient supply but also from hitherto unknown PKU-related metabolic disturbances. Further research on desaturases’ activity in PKU is warranted.

Conflicts of interest

The authors report no conflicts of interest.

Acknowledgments

We are grateful to the phenylketonuria patients and their families for their cooperation.

SDC was a recipient of a fellowship from the NUTRICIA Research Foundation at the Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical Centre, Munich, Germany. SDC would like to thank Dr Hans Demmelmair for his supervision during the fellowship and cooperation.

Acknowledgments of Financial Support

The study was supported by a grant from Poznań University of Medical Sciences (No 502-01-0103115-07588).

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