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Clinical Nutrition xxx (xxxx) xxx

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Contents lists available at ScienceDirect 56
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Clinical Nutrition 59
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journal homepage: http://www.elsevier.com/locate/clnu 61
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Narrative Review 64
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1 Dietary protein and protein substitute requirements in adults with 66
2 67
3 Q7 phenylketonuria: A review of the clinical guidelines 68
4 69
5 Q6 Sarah Firman a, b, c, *, Oliver C. Witard a, d, Majella O'Keeffe a, e, 1, Radha Ramachandran c, 1 70
6 a 71
Department of Nutritional Sciences, King's College London, London, United Kingdom
7 b 72
Department of Nutrition and Dietetics, Guy's and St Thomas' NHS Foundation Trust, London, United Kingdom
8 c
Adult Inherited Metabolic Diseases, Guy's and St Thomas' NHS Foundation Trust, London, United Kingdom 73
9 d
Centre for Human and Applied Physiological Sciences, King's College London, London, United Kingdom 74
e
10 School of Public Health, University College Cork, Western Gateway Building, Western Road, Cork, Ireland
75
11 76
12 77
13 a r t i c l e i n f o s u m m a r y 78
14 79
15 Article history: Lifelong dietary adherence is recommended in the management of phenylketonuria (PKU). Accordingly,
Received 10 September 2020
80
16 an increasing adult population require age-specific PKU guidelines on protein requirements to support
Accepted 1 November 2020 81
17 changing metabolic demands across the lifespan. Given that protein intake for dietary management of
PKU is primarily (52e80%) derived from protein substitutes, the prescribing practice of protein sub-
82
18 Keywords: 83
stitutes must be underpinned by robust evidence. Whilst dietary guidelines for PKU management is
19 Phenylketonuria 84
evolving to incorporate adult specific protein recommendations, the scientific evidence underpinning
20 Hyperphenylalaninemia
these guidelines is currently limited. Instead, the determination of protein requirements for PKU patients 85
21 Dietary management
Protein requirements
have previously been extrapolated from estimates derived from the general healthy population, based on 86
22 arguably outdated nitrogen balance methodology. Furthermore, a compensatory factor of 20e40% has 87
Protein recommendations
23 Protein metabolism been incorporated to account for the reduced uptake and utilisation of the elemental amino acids 88
24 contained in protein substitutes. However, research informing this compensatory factor have been 89
25 conducted in younger adults, with the majority of studies in non-PKU individuals. Given extensive ev- 90
26 idence that the muscle anabolic response to ingested protein is impaired in older vs. young adults, the
91
27 validity of current dietary protein recommendations for adults and older adults with PKU has been
92
28 challenged. This narrative review aims to critically evaluate the existing scientific evidence underpinning
current guidelines on protein requirements for adult PKU patients, highlighting existing gaps in
93
29 94
knowledge and directions for future research. We argue that current guidelines on protein requirements
30 95
need updating to optimise long-term physical and functional outcomes in older adults with PKU.
31 © 2020 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. 96
32 97
33 98
34 99
35 100
36 101
37 1. Introduction Europe, the majority of PKU cases are diagnosed during neonatal 102
38 screening and prevalence rates range from 1:3000 to 1:30,000 103
39 Phenylketonuria (PKU; OMIM 261600) is an autosomal recessive births [1]. With early diagnosis and treatment initiated in the 104
40 inborn error of protein metabolism resulting from a deficiency in neonatal period, individuals with PKU no longer risk developing 105
41 the hepatic enzyme, phenylalanine hydroxylase, which converts profound and irreversible intellectual disability. However, subop- 106
42 phenylalanine into tyrosine. The deficiency in phenylalanine hy- timal phenylalanine control in childhood and adulthood has been 107
43 droxylase leads to an accumulation of phenylalanine in the blood shown to lead to attention deficit, mood disturbance and impaired 108
44 and brain which, if left untreated, results in irreversible intellectual executive function [2e4]. Furthermore, the long-term effects of 109
45 disability, microcephaly, seizures and behavioural problems. In high phenylalanine levels on morbidity and ageing are yet to be 110
46 determined [5]. 111
47 Despite advances in the pharmacological management of PKU 112
48 (eg. Sapropterin dihydrochloride [BH4] or Pegvaliase), dietary 113
* Corresponding author. St Thomas' Hospital, 3rd Floor Becket House, 1 Lambeth
49 Palace Road, London, SE1 7EU, United Kingdom. management remains the mainstay of treatment. The dietary 114
50 E-mail addresses: sarah.firman@gstt.nhs.uk, sarah.firman@kcl.ac.uk (S. Firman). management of PKU was first introduced by Horst Bickel and col- 115
1
51 Majella O'Keefe and Radha Ramachandran contributed equally to this article leagues [6] in 1953 and since then dietary guidelines in children 116
and share final authorship.
52 117
53 118
https://doi.org/10.1016/j.clnu.2020.11.003
54 0261-5614/© 2020 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. 119

Please cite this article as: S. Firman, O.C. Witard, M. O'Keeffe et al., Dietary protein and protein substitute requirements in adults with
phenylketonuria: A review of the clinical guidelines, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2020.11.003
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1 with PKU have evolved. Dietary guidelines for the management of health outcomes [18,19]. Throughout this review, close consider- 66
2 adults with PKU have also developed, with the emergence of the ation was given to the methodology used to establish the protein 67
3 recommendation for lifelong adherence to the diet [7]. Notwith- requirement, including the provision of protein substitutes, and 68
4 standing, variations in dietary management guidelines during whether these methods are transferable to PKU patient 69
5 childhood and adulthood exist both within and between countries populations. 70
6 [8e12]. 71
7 Protein requirements for PKU patients are typically met through 3. The evolution of protein requirement guidelines in adults 72
8 a combination of protein containing foods in the habitual diet and with phenylketonuria 73
9 commercially available protein substitutes. Protein substitutes 74
10 constitute the majority (52e80%) of protein intake in most PKU In 1993, the United Kingdom (UK) Medical Research Council 75
11 patients [10,13]. Existing guidelines on protein requirements for (MRC) Working Party on PKU first introduced the concept that a 76
12 PKU patients have been extrapolated from estimations of protein phenylalanine restricted diet should continue into adult life [7]. 77
13 requirements for the general healthy population using nitrogen However, the oldest age group included in these guidelines was 78
14 balance methodology, with a compensatory factor accounting for children >2 years and the recommendation was to consume a 79
15 the reduced utilisation of elemental amino acids from ingested protein substitute at a dose of 2 g/kg body mass/day. Prior to this 80
16 protein substitutes [9,10,14]. However, a paucity of studies have publication, dietary guidelines for PKU management only refer- 81
17 directly compared and contrasted estimations of protein re- enced data on phenylalanine restriction in children [20,21]. Hence, 82
18 quirements between PKU patients and the general population while the importance of age-specific protein guidelines for PKU 83
19 across the lifespan. With growing consensus for lifelong PKU di- patients was recognised, the MRC report was unable to make 84
20 etary management [7e10,15], there is now an increasing adult and explicit reference to data on protein requirements tailored to ado- 85
21 ageing population who require age specific guidelines for protein lescents or adults with PKU. Indeed, it was not until the revised 86
22 requirements to support protein needs across their lifespan. American College of Medical Genetics and Genomics (ACMG) 87
23 Accordingly, the main purpose of this narrative review is to criti- practice guidelines in 2014 [8,15,22] that protein requirement 88
24 cally evaluate the scientific evidence underpinning current clinical guidelines specific to adults with PKU finally emerged (see Table 1). 89
25 guidelines for protein requirements in adult PKU patients, with a These guidelines recommended a protein intake equivalent to 90
26 view to highlighting existing gaps in knowledge for future research. 120e140% of the RDA [23] for age, and therefore take into consid- 91
27 eration the changes in protein requirements as individuals age from 92
28 4 years into adulthood. Prior to the ACMG guidelines, a review by 93
29 2. Methods MacLeod & Ney [24] on the nutritional management of PKU 94
30 concluded that insufficient data existed to devise evidence-based 95
31 MEDLINE and EMBASE was used to search for published litera- protein recommendations for PKU patients over 19 years old. 96
32 ture on protein requirement guidelines for PKU patients across the Subsequently, in 2016, Singh et al. [25] published an expanded 97
33 lifespan (see Box 1). Relevant references from articles retrieved, and review and expert consultation on PKU nutrition therapy guidelines 98
34 conference abstracts and literature known to authors also were that advanced the 2014 ACMG practice guidelines. Importantly, 99
35 reviewed. This review classifies adults as
18 years and older adults guidelines on protein requirements for those 4þ years contrasted 100
36 as
60 years. The Recommended Dietary Allowance (RDA) or with the 2014 guidelines in that the 120e140% of age appropriate 101
37 Recommended Dietary Intake (RDI) for protein are commonly re- 2005 dietary recommended intakes [26] were used rather than the 102
38 ported in adult protein requirement guidelines (Table 2), herein 1989 RDA [23]. For those PKU patients aged 18 years, the 2005 103
39 referred to as RDA. The RDA is defined as meeting the protein needs guidelines were lower than the previous 1989 RDA; however, for 104
40 of 97.5% of healthy individuals and is considered to be the ‘safe those >18 years of age, no differences in protein recommendations 105
41 level’ for protein intake by the FAO/WHO/UNU reports. existed (see Table 2). 106
42 While the definition of protein requirement may be considered The most recent (2017) European PKU guidelines draw on pre- 107
43 controversial, for the purpose of this review, the protein require- viously published guidelines and survey data of protein re- 108
44 ment refers to the metabolic demand and the efficiency of protein quirements used in clinical practice across European countries for 109
45 utilisation, as detailed by Millward [16]. Specifically, the metabolic adults with PKU [9]. For the majority of countries, no data were 110
46 demand relates to what the organism needs in terms of amino acids collected on protein prescribing practices in adult PKU patients. 111
47 for maintenance plus additional needs for growth, pregnancy and Whilst the ACMG 2014 [8,15,22] and updated 2016 GMDI/SERC [25] 112
48 lactation; and efficiency of protein utilisation describes the rela- guidelines recommend that PKU patients consume a protein intake 113
49 tionship between dietary protein intake and satisfaction of meta- of 120e140% of age-specific protein RDA [23], this European 114
50 bolic demands. Protein requirements for the general population are consensus guideline recommends 140% of FAO/WHO/UNU 2007 115
51 typically based on nitrogen balance studies [17], and in essence age-specific protein requirements [27]. Furthermore, the guideline 116
52 equates to the minimum dietary protein intake necessary to bal- specifies that the additional 40% of dietary protein is prescribed in 117
53 ance nitrogen losses from the body. In contrast, for the purpose of the form of L-amino acid supplements, a 20% compensatory factor 118
54 this review, a protein recommendation is defined as the protein to account for the digestibility and utilisation of amino acids from 119
55 intake that serves to optimise metabolic function and improve the supplement, and a further 20% compensation to optimise 120
56 phenylalanine control [9]. 121
57 Alongside the European PKU guideline9, the 2017 Australasian 122
58 Box 1 consensus guidelines28 for those aged 18þ years recently set their 123
59 Search terms protein requirement guideline at the 140% of the US and European 124
60 PKU dietary management guidelines of age appropriate RDI. The 125
61 Australasian guidelines reference the Nutrient Reference Values 126
62 from the Australia and New Zealand 2006 report [29], which 127
phenylketonuria* OR PKU OR hyperphenylalaninemia
63 AND management OR guidelines OR consensus included an increased protein requirement for adults >70 years. 128
64 The Australasian guidelines for protein requirements in adults with 129
65 PKU reflects this viewpoint and advocate an increased protein 130
2
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1 Table 1 66
Summary of clinical phenylketonuria (PKU) guidelines that include protein recommendations for adults with PKU. Q5
2 67
3 Author/date Population Protein recommendation Evidence cited for adult PKU protein requirements 68
4 MacLeod & Ney (2010) Lifespan 15e18 years: 2.0 g/kg/day Metges CC et al. (2000)
69
5 [Narrative review]
19 years: NA Dangin M et al. (2001) 70
6 van Rijn M et al. (2007) 71
7 Camp et al. (2014) United >4 years to adults: Recommended Dietary Allowances (1989) 72
[Conference proceedings] States 120e140% RDA for age
8 73
Lifespan
9 Singh et al. (2014) [Guideline] United >4 years to adults: Recommended Dietary Allowances (1989) 74
10 States 120e140% RDA for age Metges CC et al. (2000) 75
11 Lifespan Gropper SS & Acosta PB (1991) 76
12 Vockley et al. (2014) United >4 years to adults: Cited guideline by Singh et al. (2014) 77
[Guideline] States 120e140% RDA for age
13 Lifespan
78
14 Singh et al. (2016) [Guideline] United >4 years to adults: Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, 79
15 States 120e140% DRI for age Cholesterol, Protein, and Amino Acids (2005) 80
16 Lifespan Gropper SS & Acosta PB (1991) 81
van Wegberg et al. (2017) Europe FAO/WHO/UNU 2007 plus Protein and amino acid requirements in human nutrition: report of a joint FAO/WHO/
17 82
[Guideline] Lifespan additional 40% from L-amino UNU expert consultation (2007)
18 acids supplements Metges CC et al. (2000) 83
19 Gropper SS & Acosta PB (1991) 84
20 Daenzer M et al. (2001) 85
21 Monch E et al. (1996) 86
Hennermann JB et al. (2013)
22 Schindeler S et al. (2007)
87
23 Hidalgo IJ & Borchardt RT (1990) 88
24 Matalon R et al. (2007) 89
25 Matalon R et al. (2006) 90
Inwood et al. (2017) Australasia 10e18 years: Nutrient reference values for Australia and New Zealand (2006)
26 91
[Guideline] Lifespan 1.0e1.5 g/kg/day Cited guideline by Singh et al. (2016)
27 >18 years: Cited guidelines by van Spronsen et al. (2017) 92
28 140% RDA for age 93
29 94
30 95
31 96
32 Table 2 97
Protein requirements for the general population (g/kg body mass/day) used to inform the development of PKU dietary management guidelines.
33 98
34 Age FAO/WHO/UNU (1985)a RDA (1989)b DRI (2005)c RDA values Aus/NZ RDI (2006)d FAO/WHO/UNU (2007)e 99
35 Male 100
36 18 years 0.86 0.80 0.85 0.99 0.85 101
37 Female 102
18 years 0.81 0.80 0.85 0.77 0.82
38 103
Male
39 19e30 years 0.75 0.80 0.80 0.84 0.83 104
40 31e50 years 0.75 0.80 0.80 0.84 0.83 105
41 51e70 years 0.75 0.80 0.80 0.84 0.83 106
>70 years 0.75 0.80 0.80 1.07 0.83
42 107
Females
43 19e30 years 0.75 0.80 0.80 0.75 0.83
108
44 31e50 years 0.75 0.80 0.80 0.75 0.83 109
45 51e70 years 0.75 0.80 0.80 0.75 0.83 110
46 >70 years 0.75 0.80 0.80 0.94 0.83 111
47 a
Food and Agricultural Organization: World Health Organization: United Nations. Energy and protein requirements. Report of a joint FAO/WHO/UMA Expert Consultation. 112
48 Technical Report Series No. 724. Geneva: World Health Organization, 1985. 113
b
National Research Council Recommended Dietary Allowances. 10th ed. Washington, DC: National Academies Press; 1989.
49 c
114
Institute of Medicine. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids. Washington, DC: The National
50 Academies Press; 2005.
115
51 d
National Health and Medical Research Council. Nutrient reference values for Australia and New Zealand including recommended dietary intakes. Canberra: NHMRC; 2006. 116
e
52 Joint FAO/WHO/UNU Expert Consultation on Protein and Amino Acid Requirements in Human Nutrition (2002: Geneva, Switzerland), Food and Agriculture Organization of 117
53 the United Nations, World Health Organization & United Nations University. Protein and amino acid requirements in human nutrition: report of a joint FAO/WHO/UNU expert 118
consultation. World Health Organization; 2007.
54 119
55 120
56 121
57 122
58 requirement for the elderly PKU population.28 To this end, the evidence-based protein recommendations to meet their metabolic 123
59 Australasian consensus is the only guideline with increased protein demand is essential. In terms of progress, protein requirements for 124
60 requirements for older patients. adults were initially devoid, but more recently, guidelines have 125
61 With the recent initiative to continue dietary management for developed to incorporate protein requirements specific to adults 126
62 PKU patients into adult life, tailoring protein guidelines to older with PKU, as outlined in Table 1. However, these recommendations 127
63 adults with PKU is crucial to the lifelong health and well-being of are extrapolated from the general healthy population with an 128
64 this patient group. Moreover, given that modifying the source of additional compensatory factor of 20e40% and the scientific evi- 129
65 protein consumed is integral to phenylalanine control, establishing dence underpinning these guidelines warrants further review. 130
3
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1 4. Determining protein requirements in adults with Protein recommendations for PKU patients also must consider 66
2 phenylketonuria the sources of dietary protein consumed alongside the total daily 67
3 intake. Data from nitrogen balance studies consisting of animal, 68
4 4.1. Extrapolation from general population protein requirement vegetable or mixed proteins were used to inform the most recent 69
5 estimations FAO/WHO/UNU 2007 [27] RDA for protein in the general healthy 70
6 population. Whilst vegetable and fruit protein constitute a portion 71
7 The majority of studies used to inform national and interna- of protein intake for those with PKU, animal proteins would be 72
8 tional guidelines on protein requirements for adults and older devoid for the majority. Protein substitutes consisting of elemental 73
9 adults in the general population utilise data generated using amino acids or GMP provide the major protein source for most PKU 74
10 nitrogen balance methodology [17,23,26,27,30]. A more patients [10,13]. Due to the digestibility and bioavailability profile of 75
11 contemporary and valid approach to estimating protein re- vegetable- and fruit-based protein, and the utilisation of protein 76
12 quirements is the Indicator Amino Acid Oxidation (IAAO) substitutes, the protein RDA for the general healthy population 77
13 method [31]. Using this method, Courtney-Martin and colleagues could not be generalised to the PKU population. Accordingly, recent 78
14 [32] reported that protein requirements for healthy adults across PKU guidelines advocate exceeding protein requirements for the 79
15 the lifespan were greater than previously published [17]. general population by 20e40% to account for reduced utilisation of 80
16 Accordingly, the use of nitrogen balance studies in older adults amino acids and to optimise circulating phenylalanine levels. 81
17 has been challenged given that older adults have been shown to However, utilising IAAO methodology, Turki et al. [42] reported 82
18 adapt to a lower protein intake by breaking down lean tissue protein requirements in four children (9e18 years of age) with mild 83
19 mass to meet nitrogen equilibrium [33]. Therefore, it may be hyperphenylalanemia to be 1.85 g/kg body weight/day which could 84
20 argued that the protein RDA, based on nitrogen balance data, is be 39e55% higher than the upper end of the current recommen- 85
21 inadequate to meet the changing metabolic demands of adults dation for PKU patients, depending on age. A study is currently 86
22 for maintaining fat-free mass and functional capacity [33e36]. underway to determine protein requirements from L-amino acid vs. 87
23 Furthermore, this definition does not account for different health GMP protein substitutes in adults with PKU utilising the IAAO 88
24 conditions and specialist dietary modifications, such as those method [43]. However, to date, no published studies have utilised 89
25 experienced by PKU patients. As detailed above, adult specific the IAAO approach to determine protein requirements in adult PKU 90
26 PKU protein guidelines have evolved since the publication of patients. 91
27 MacLeod & Ney [24] to incorporate age appropriate guidelines 92
28 from a range of reports (see Table 1). However, these reports 4.2. Amino acid uptake and utilisation in PKU patients 93
29 have been established for the general healthy adult population 94
30 [17,23,26,27,29,30], as summarised in Table 2, and do not account Several studies referenced in the PKU dietary guidelines provide 95
31 for sub-populations with health conditions such as PKU. evidence for the difference in utilisation of elemental amino acids 96
32 Current guidelines for protein requirements in PKU patients versus whole protein, as detailed in Table 1. Specifically, Gropper 97
33 utilise guidelines set for the general population and are age-specific et al. [44] compared plasma amino acid profiles and urea nitrogen 98
34 until 19 years, after which protein requirements remain consistent content in 10 healthy male volunteers (age range 18.5e33.9 years) 99
35 across the adult lifespan (Table 2). Given that age-related changes who consumed a test meal of either cottage cheese (whole protein), 100
36 in metabolic and physiological needs of the musculoskeletal system L-amino acids matched in composition to cottage cheese, or a 101
37 exist, concerns have been raised regarding how appropriate a “one mixture of cottage cheese and L-amino acids. The transient rise in 102
38 size fits all” approach to devising protein requirements for PKU plasma amino acid (total and essential amino acids) concentrations 103
39 patients across the lifespan. Regarding protein requirements for was more rapid and of greater magnitude in the L-amino acids 104
40 muscle health, there is extensive evidence that the muscle anabolic groups vs. the whole protein group. Moreover, amino acid con- 105
41 response to ingested dietary protein is impaired in older adults centrations returned to baseline levels more rapidly with ingestion 106
42 compared with their younger counterparts [37,38]. This phenom- of L-amino acids. In contrast, no difference in plasma urea nitrogen 107
43 enon has been termed muscle anabolic resistance. Accordingly, content was observed between test meals. These data provide 108
44 increasing dietary protein intake has been advocated as an insight into the impact of ingesting L-amino acids on plasma amino 109
45 important lifestyle strategy to overcome age-related anabolic acid kinetics and warrants a follow up study in PKU patients, 110
46 resistance and by extension ameliorate the loss of skeletal muscle particularly when combined with other measurements of whole- 111
47 mass associated with advancing age [33e37]. It follows that body protein metabolism. 112
48 consideration has been given to increasing protein requirements of Several experimental studies also provide insight into the 113
49 older adults, as evidenced by the Nutrient Reference Values for metabolic impact of a more rapid and greater rise in plasma amino 114
50 Australia and New Zealand 2006 report [29] that recommends a acid availability on protein synthesis rates [45e48]. For instance, 115
51 25% increase in protein intake for adults >70 years of age. Support Metges et al. [45] compared the uptake and utilisation of labelled 116
52 for this initiative stems from studies demonstrating that protein leucine with the ingestion of an isolated whole protein (casein 117
53 intakes equivalent to the RDA (0.8 g/kg body weight/day) are derived from goats milk) source vs. a free amino acid mixture that 118
54 inadequate to meet metabolic demands for older adults [39,40]. In simulated the amino acid composition of casein in 14 healthy 119
55 this regard, Campbell et al. [39] reported a significant reduction in young adult volunteers (mean age range of 20.6e26.8 years). 120
56 mid-thigh muscle area and an associated decrease in urinary ni- Although study diets were isocaloric, isonitrogenous and matched 121
57 trogen excretion in 10 healthy participants (aged 55e77 years) who for leucine content, the ingestion of casein (whole protein) was 122
58 adhered to a protein intake of 0.8 g/kg body weight/day over 14 associated with lower leucine oxidation rates, and increased non- 123
59 weeks. The Nutrient Reference Values derived from the Australia oxidative leucine disposal (NOLD) and net protein synthesis rates, 124
60 and New Zealand 2006 report informs the Australasia PKU protein compared with free amino acids. Taken together, these data [44,45] 125
61 guideline [28]. With the exception of this PKU protein guideline, suggest that the increase in plasma amino acid concentrations with 126
62 other protein recommendations for adults with PKU remain free amino acids is of greater magnitude and more rapid than intact 127
63 consistent across the lifespan and therefore are not likely adequate protein sources. Thus, amino acid oxidation rates are higher and 128
64 to meet the metabolic and physiological needs associated with protein retention lower when consuming free amino acids vs. a 129
65 ageing [41]. whole protein source. Whilst these studies are cited in current 130
4
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1 guidelines, the rationale that protein requirements of PKU patients indicating a faster transit through the stomach compared to casein 66
2 exceeds the general population by 20e40% remains unclear. In this protein. While this study provides useful mechanistic insight into 67
3 regard, these studies fail to provide insight into whether findings differences in amino acids kinetics between amino acid sources, 68
4 would differ if additional amino acids were provided to those follow up clinical studies are warranted in PKU patients. 69
5 consuming free amino acids to compensate for the differences in As further insight into the impact of protein source on amino 70
6 protein metabolism when compared to whole protein. Further- acid uptake and utilisation in PKU patients, Dangin et al. [47] 71
7 more, these studies were conducted in healthy adults and the test conducted a study in 22 healthy male volunteers (aged 25 ± 1 72
8 diets did not reflect what is typically consumed by a PKU patient, years) where ‘slowly’ digested proteins resulted in improved 73
9 thus limiting the translation of findings to PKU patients. postprandial protein retention rate compared to ‘fast’ digested 74
10 To our knowledge, the review by MacLeod & Ney [24] is the only protein. These data suggested that protein digestibility affects 75
11 report to cite a study by van Rijn et al. [49] that investigated rates of amino acid utilisation, independent of amino acid composition. To 76
12 protein utilisation in young adults with PKU (mean age of 27 ± 7 test this theory, human participants were infused with L-[1e13C] 77
13 years) vs. healthy young adults (mean age of 32 ± 4 years). In this leucine and then consumed one of the following 30g protein meals: 78
14 study, healthy volunteers consumed the protein RDA (0.8g protein/ casein, free amino acids simulating the amino acid composition of 79
15 kg/day) from standard protein sources (67% milk protein and 33% casein, whey proteins, or repeated meals of whey proteins. 80
16 vegetable protein) whereas PKU patients consumed protein pre- Consistent with previous study findings [45e48], a more profound 81
17 scribed to meet RDA from dietary protein allowance (0.1e0.2 g and transient rise in plasma leucine concentrations and higher 82
18 protein/kg per day) and amino acid supplements, plus an additional oxidation rate was observed in the free amino acid group. When 83
19 compensatory 20% from amino acid supplements. In tracing the comparing the intake of free amino acids (‘fast protein’) with a 84
20 fate of labelled L-[1e13C]-valine to quantify amino acid uptake and whey protein test drink consumed in small doses to mimic a slower 85
21 utilisation, this study revealed no differences in whole-body pro- digestion rate (‘slow protein’), free amino acids moderately reduced 86
22 tein turnover, amino acid oxidation and net protein balance be- postprandial rates of protein breakdown whereas the whey protein 87
23 tween groups after consuming their respective diets. This study is group exhibited a more profound and sustained inhibition of pro- 88
24 the first to demonstrate that the recommendation for adult PKU tein breakdown. These data raise an interesting practical consid- 89
25 patients to consume the protein RDA plus an additional 20% from eration for PKU patients regarding whether the consumption of 90
26 amino acid supplements resulted in a comparable response of protein substitutes (‘fast’ protein source) could mimic a slower 91
27 whole-body protein metabolism to healthy participants consuming digested protein if consumed in smaller repeated doses. While 92
28 the protein RDA (0.8 g/kg/day). Moreover, these data highlight that current guidelines recommend the spacing of protein substitutes 93
29 for adult PKU patients following a PKU diet, protein requirements evenly throughout the day, the implications of recommending 94
30 should include a minimum increment of 20% from protein sub- more frequent smaller protein doses need to be balanced against 95
31 stitutes to be comparable to the protein requirements of their the inherent practical difficulties. Furthermore, in terms of long- 96
32 healthy counterparts. term health, meeting the protein threshold by provision of 97
33 The most recent European PKU consensus guidelines [9] adequate per-meal protein dosing presents a promising strategy for 98
34 recommend that ingested L-amino acids should be distributed as reducing age-associated muscle loss [53], and warrants consider- 99
35 three or more equal portions throughout the day, equating to ~20g ation when recommending protein substitute dosing and fre- 100
36 of protein equivalent per serving for adults. This guideline cites a quency for adult and ageing PKU patients. 101
37 study by Monch et al. [50] that investigated the metabolic effects of The majority of studies demonstrate that amino acid uptake and 102
38 different doses of L-amino acids using healthy volunteers and PKU utilisation is reduced with free amino acids compared to equal 103
39 patients. In healthy participants (aged 26e46 years), multiple bo- intakes of whole proteins. With the exception of van Rijn et al. [49], 104
40 luses, compared to a single bolus, resulted in a markedly reduced studies administered matched protein quantities and amino acid 105
41 appearance of amino acids into the circulation, as well as lower composition, and therefore fail to elucidate whether provision of an 106
42 urea and insulin concentrations. Consistent with this observation, increased free amino acid dose improves net protein synthesis 107
43 urinary nitrogen studies in PKU patients (9 with classical PKU and 1 rates. It is unclear why the study by van Rijn et al. [49] has not been 108
44 with hyperphenylalaninemia) reported higher nitrogen excretion cited by all recent PKU dietary management guidelines as evidence 109
45 rates when L-amino acids were administered in two doses for the recommendation to increase the protein RDA by 20%. A 110
46 (6.3e12.4g/24 h) than three doses (4.7e10.8g/24 h). Whilst this limitation of all studies investigating amino acid uptake and uti- 111
47 study provided insight into the biochemical and metabolic lisation relates to the tightly controlled nature of laboratory con- 112
48 response after consumption of L-amino acids in PKU patients, the ditions with trial meals provided over a maximum of 24 h, thus not 113
49 sample sizes were small and no statistical analysis was reported reflecting the diets and lifestyles of free-living adults with PKU. 114
50 which limits the inference of findings. Furthermore, all studies are limited by a small sample size and were 115
51 Protein digestibility is recognised as an important factor when conducted in young healthy adults. It also has been suggested that 116
52 considering amino acid requirements [51]. Protein requirement older adults may exhibit a greater amino acid utilisation with ‘fast’ 117
53 guidelines for PKU patients refer to studies that measured the absorbed proteins compared to younger adults [46], and therefore 118
54 metabolic fate of ‘fast’ (i.e., L-amino acids and whey protein) vs. ‘slow’ the findings may not be directly applicable to older adults. Whilst 119
55 (i.e. casein and whey ingested in repeat doses) digestible protein further research and long-term studies are warranted to draw any 120
56 sources [47,52]. Using a rodent model, Daenzer et al. [52] investigated definitive conclusions, these data raise an interesting consideration 121
57 the metabolic fate of L-amino acids compared to casein when a 9-day for ageing PKU patients whose protein intake could predominately 122
58 adaption period with standardised meals was followed on day 10 consist of elemental amino acids. Moving forward, future studies 123
59 with the administration of either a single meal of 13C-labeled casein should be designed to determine the impact of modifying amino 124
60 or an amino acid mixture containing labelled leucine and lysine. acid kinetics with the ingestion of protein substitutes on rates of 125
61 Consistent with previous findings [45], higher urinary nitrogen whole-body and muscle protein synthesis in PKU patients. This 126
62 excretion rates were observed with the ingestion of L-amino acids vs. information will be important in establishing the metabolic and 127
63 casein protein, indicating reduced net protein synthesis. Further- physiological needs of PKU patients and will help inform protein 128
64 more, the rate of appearance of free 13C-leucine into the intestinal recommendations for optimal metabolic function in this popula- 129
65 mucosa amino acid pool was greater with the L-amino acids, tion across the lifespan. 130
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1 There is a common misconception that high protein intakes 5. Practical implications 66

2 (i.e. 2-3 times the RDA) are harmful to kidney function in other- 67
3 wise healthy individuals [54,55]. However, a recent review of co- National and international guidelines on protein requirements 68
4 morbidity in PKU patients highlighted that kidney function may for the general healthy population form the basis of protein re- 69
5 be impacted by the high protein intake from amino acid supple- quirements in adults with PKU. However, there is ongoing debate 70
6 ments [5]. Consistent with this review and the European PKU around whether “one size fits all” for protein requirements in all 71
7 consensus guidelines [9], a cross-sectional study of 67 PKU pa- adult and ageing populations given the age associated changes in 72
8 tients (aged 15e43 years) demonstrated a reduction in glomer- metabolic and physiological demands, particularly with regards to 73
9 ular filtration rate (GFR) in those individuals with dietary protein the musculoskeletal system. The protein RDA is based on the pro- 74
10 and L-amino acid intakes in excess of the RDA over an extended tein intake required to meet nitrogen equilibrium rather than 75
11 period (i.e. years) [56]. However, the reduction in GFR remained optimal health outcomes and does not consider long-term physical 76
12 within the target physiological range. Although proteinuria was and functional outcomes. For the general population, higher pro- 77
13 detected in 31% of PKU patients, no control group was included in tein intakes have been suggested to overcome age-related muscle 78
14 the study and protein intakes were measured at a single-time anabolic resistance [37]. However, when devising recommenda- 79
15 point, thus limiting the strength of findings from this study. tions across the lifespan, multiple factors need to be considered, 80
16 With guidelines on protein requirements for adult PKU patients including physical activity level, type of protein intake (fast versus 81
17 exceeding the protein RDA, longitudinal studies are warranted to slow protein), distribution of protein over the day and energy 82
18 determine if any negative health outcomes, including kidney intake, all of which need consideration for PKU patients. 83
19 function, are associated with higher protein intakes in PKU The most recent PKU guidelines recommend that protein in- 84
20 patients. takes exceed the RDA for age by 20e40%. This incremental factor 85
21 serves to compensate for the reduced uptake and utilisation of 86
22 amino acids from protein substitutes, which constitute the majority 87
23 4.3. Plasma phenylalanine control with higher doses of protein of protein intake in individuals on a PKU diet. As detailed above, the 88
24 substitutes evidence underpinning this notion is primarily based on inference 89
25 from relatively small-scale experimental studies conducted in 90
26 The measurement of phenylalanine control discussed below healthy young adults that compare the metabolic fate of free amino 91
27 refers to circulating phenylalanine concentrations, unless other- acids vs. whole protein sources. Accordingly, it is unclear from these 92
28 wise specified. The role of protein substitutes in reducing plasma studies whether this incremental factor is adequate to meet the 93
29 phenylalanine concentrations is well established, as evidenced by potential increased needs of older adult PKU patients. 94
30 recent European consensus guidelines [9]. These guidelines were 95
31 the first to recommend an incremental factor for protein sub- 96
32 stitutes in an attempt optimise phenylalanine control. Although 6. Future research directions 97
33 studies in children with PKU report improvements in phenylalanine 98
34 control with higher intake of protein substitutes [57], a limited With inherited metabolic disease services now providing guid- 99
35 number of studies demonstrate the benefits of increasing intakes of ance on dietary management for PKU patients across the adult 100
36 protein substitutes on phenylalanine control adults PKU patients. In lifespan, future research is urgently needed to establish evidence- 101
37 this regard, Duran et al. [58] demonstrated reduced plasma based protein requirements and recommendations to overcome 102
38 phenylalanine levels in five maternal PKU patients who received age-related anabolic resistance and prevent skeletal muscle mass 103
39 hospitalisation to support adhere to their prescribed protein sub- associated with advancing age in PKU patients. Guidelines on 104
40 stitute dose. However, the impact of a 20% increase in provision of protein requirements should consider the metabolic demands of 105
41 protein substitute on phenylalanine concentrations in adult PKU PKU patients across the adult lifespan and the efficiency of protein 106
42 patients remains unclear from these studies and warrants further substitutes to meet their metabolic needs. Factors influencing 107
43 investigation. protein metabolism such as the dose of protein substitute ingested, 108
44 Sufficient dietary protein intake is a pre-requisite for protein daytime distribution of protein substitutes, digestibility of protein 109
45 anabolism and may be linked with improvements in phenylala- sources and energy intakes, and the impact these factors have at 110
46 nine control observed with higher doses of L-amino acids. How- different ages, warrants further investigation in a PKU population. 111
47 ever, it has also been suggested that the intake of large neutral Historically, protein requirements for the general population are 112
48 amino acid (LNAA) leads to reduced plasma phenylalanine con- determined by the outdated nitrogen balance technique and have 113
49 centrations, as mediated by an alteration in the transportation of been extrapolated to PKU patients. However, recent studies utilis- 114
50 phenylalanine across the intestinal mucosa. Consistent with this ing the more advanced method of IAAO suggest that protein re- 115
51 notion, Hidalgo et al. [59] conducted a study utilising Caco-2 cell quirements should be increased in both the general population and 116
52 monolayer and demonstrated that high intakes of cationic L- PKU patients. Furthermore, establishing biochemical and functional 117
53 amino acids and large neutral amino acids reduced phenylalanine markers that represent protein status in adult PKU patients will 118
54 uptake in the gut. Studies in human PKU patients [60,61] and improve the monitoring and management of PKU, allowing for a 119
55 mouse models [60] also reported a >50% decrease in plasma more individualised approach to determining protein requirements 120
56 phenylalanine concentrations with the intake of LNAA, with PKU in this patient group. 121
57 patients consuming 0.5e1.0 g/kg/day of a LNAA formula for 7 122
58 days. In contrast, Schindeler et al. [62] reported minimal benefits 123
59 on plasma phenylalanine concentrations in PKU patients that Synopsis 124
60 consumed either LNAA (250 mg/kg/day) or placebo along with 125
61 patients that continued with their habitual protein substitute or Despite the recommendation for lifelong dietary adherence in 126
62 no protein substitute. Of note, the dose of LNAA administered in the management of phenylketonuria (PKU), this review highlights 127
63 this study was lower than previous studies [60,61]. The role of that protein requirements presented in their current form may not 128
64 LNAA in altering phenylalanine uptake is also of relevance at the adequately offset age-related changes in protein metabolism across 129
65 blood brain barrier [60e62]. the adult lifespan for PKU patients. Q1 130
6
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