in phenylketonuria ( pku , omim 261600 ) , dietary restriction of phenylalanine ( phe ) has been the cornerstone of treatment for over 50  years . the result of dietary treatment is a near normal cognitive outcome , although mild neuropsychological disturbances may still occur ( christ et al . large neutral amino acids ( lnaas ) have been suggested as an alternative treatment to further improve outcome . 2006 ) , augmentation of cerebral neurotransmitter synthesis ( lou et al . 1985 ; gttler and lou 1986 ; lou et al . 1987 ; lykkelund et al . 1988 ) , and/or elevation of brain non - phe lnaa concentrations ( van spronsen et al . , we summarize the reports of the clinical trials of lnaas and the rationales behind the use of lnaas . apart from phe , lnaas include tyrosine , tryptophan , threonine , methionine , valine , isoleucine , leucine , and histidine ( van spronsen et al . 2009 ) . in healthy individuals , lnaa treatment as an alternative to dietary phe restriction was suggested as early as 1948 ( christensen et al . 1948 ) and first studied in rats in 1976 ( andersen and avins 1976 ) . since then , different combinations of lnaas have been designed , based on different rationales and treatment targets . the underlying rationale for this therapeutic strategy is that elevated brain phe concentrations are considered to be neurotoxic . because all lnaas share a common transport system in order to enter the brain , and high plasma concentrations of lnaas may block the transport of phe into the brain ( pardridge 1998 ) , increasing blood lnaa concentrations may reduce uptake of phe into the brain . lnaa - treated hyperphenylalaninemic rats were shown to have significantly reduced brain phe concentrations compared to untreated hyperphenylalaninemic control rats , at similar blood phe concentrations ( andersen and avins 1976 ) . later studies in pku patients showed that supplementation with valine , isoleucine , and leucine resulted in slightly improved neuropsychological function in patients with pku ( jordan et al . ( 2003 ) showed that oral lnaa supplementation reduced brain phe concentrations , and improved neurophysiological and neuropsychological functioning ( pietz et al . differences in outcome may be related to composition , dosing , route of administration , and duration of the supplementation period . the rationale for this therapeutic strategy is that high plasma phe concentrations result in decreased brain neurotransmitter concentrations . this is reflected by reduced brain neurotransmitter concentrations in the pku mouse brain ( puglisi - allegra et al . 2002 , 2008 , 2009 ) , and by reduced concentrations of dopamine and serotonin and their metabolites in pku patients , both in cerebrospinal fluid ( csf ) ( butler et al . 1981 ; lou et al . 1985 ; burlina et al . 2000 ) , and in autopsied brain tissue ( mckean 1972 ) . in pku patients with an unrestricted natural protein intake , tyrosine and tryptophan supplementation have been shown to improve neurotransmitter metabolism , reaction time , and vigilance ( gttler and lou 1986 ; lou et al . 1988 ) , suggesting that dietary treatment could be replaced by a combination of tyrosine and tryptophan . further support for such a treatment strategy was based on the theory of prefrontal lobe dysfunction ( diamond et al . this theory presumes that the prefrontal cortex is most affected by dopamine depletion , because dopaminergic neurons innervating the prefrontal cortex have relatively high levels of activity and higher dopamine turnover , inducing dopamine - dependency . following these lines of consideration , large doses of tyrosine or l - dopa were expected to have a positive effect . however , later studies with large doses of tyrosine or l - dopa did not show positive results ( lou 1994 ; mazzocco et al . this therapeutic strategy of supplementing lnaa is based on the rationale that lnaa transport not only occurs at the blood brain barrier ( bbb ) , but also at the gut  blood barrier . after performing studies in mice , matalon et al . ( 2003 ) performed an open and a double - blinded trial in patients , who received lnaas three times daily with meals , while patients regular diet was unchanged ( matalon et al . a decrease in blood phe concentrations up to 50% of initial values was found on lnaa supplementation of 0.51.0  g kg day ( matalon et al . this suggests that lnaa supplementation superimposed on regular dietary treatment may reduce phe absorption in the gastrointestinal tract . if reduction in blood phe concentrations indeed results from reduced phe absorption , supplementation of all lnaas at this dosage may not be required , as supplementation with threonine up to 50  mg / kg is reported to be sufficient to reduce blood phe concentration by 2050% ( sanjurjo et al . in addition , the reduction of blood phe concentrations following lnaa supplementation may at least partly result from increased protein synthesis resulting from increased essential amino acid availability , in case of essential amino acid deficiency . however , it is largely unknown whether the patients studied had essential amino acid deficiencies ( due to insufficient intake of natural protein and/or protein substitute ) , as so far , none of the clinical studies on the effects of lnaa supplementation quantified the amount of natural protein and protein substitute intake patients used . finally , reduced blood phe concentrations on lnaa supplementation may just be a matter of timing of blood sampling , as suggested by prof . reduced brain lnaa concentrations have been reported in pku mice ( smith and kang 2000 ; pascucci et al . . in autopsied brain tissue of pku patients , mckean reported reduced tyrosine concentrations ( mckean 1972 ) . the rationale for lnaa supplementation to increase cerebral lnaa concentrations is , again , that lnaas share the same transport system as phe for entering the brain . where rationale a hypothesizes that it is important to decrease the brain phe concentration , rationale d hypothesizes that a deficiency in any essential amino acid in the brain may affect brain ( protein ) metabolism . consequently , rationale d focuses on the effect of lnaa treatment on brain lnaa concentrations , rather than on ( a decrease in ) brain phe concentrations . ( 2009 ) showed that a significant negative relationship exists between plasma phe concentration and cerebral protein synthesis in patients with pku . however , this finding does not differentiate between the influence of elevated brain phe concentrations and reduced brain non - phe lnaa concentrations on cerebral protein synthesis . recent trials with lnaa supplements in pku patients include lnaa alone ( pietz et al . 1999 ) , lnaa with extra lysine and/or arginine ( koch et al . 2003 ; moats et al . 2003 ; matalon et al . 2006 , 2007 ; schindeler et al . whatever combination of amino acids is given , lnaa treatment strategies start with a more or less normal ( i.e. , unrestricted ) intake of natural protein . the response of blood phe concentrations to lnaa treatment ( pietz et al . 1999 ; koch et al . 2003 ; moats et al . 2003 ; matalon et al . 2006 , 2007 ; schindeler et al . 2007 ) is comparable to the response of blood phe concentrations to the protein substitutes in the traditional dietary treatment ( macdonald et al . , we calculated prescribed lnaa intake for several treatment regimes , based on an adult pku patient with a body weight of 70  kg ( fig .  1 ) . in fig .  1 , prescribed lnaa intake is compared among four different diets : ( 1 ) conventional dietary pku treatment consisting of phe - restriction combined with synthetic amino acid mixtures , ( 2 ) lnaa supplementation ( 0.5  g kg day ) with natural protein intake at 0.8  g kg day , the recommended daily allowance ( rda ) ( national research council 1989 ) , ( 3 ) lnaa supplementation combined with conventional dietary treatment , ( 4 ) rda of protein of healthy adults . for calculation of lnaa content of synthetic amino acid mixtures ( treatment 1 ) , the mean of the following 14 amino acid mixtures was used : pku3 ( milupa ) , pku3advanta ( milupa ) , xp maxamum ( shs ) , lowphlex powder ( shs ) , phlexy-10 drinkmix ( shs ) , phlexy-10 tablets ( shs ) , pk aid-4 ( shs ) , aminogran ( ucb pharma ) , pku express powder ( vitaflo ) , phenex-2 ( ross ) , xphe advance ( metax ) , easiphen ( shs ) , pku express liquid ( vitaflo ) , lophlex liquid ( shs ) . regarding lnaa supplementation ( treatments 2 and 3 ) , a dose of 0.5  g kg day is used , based on several recent trials following the work of moats et al . prescribed lnaa intake of these treatments was calculated as the mean of lanaflex ( shs international ) and neophe ( solace nutrition ) . the dietary regimen of lnaa supplementation combined with conventional dietary treatment ( treatment 3 ) reflects the regimen studied by matalon et al . 1prescribed large neutral amino acids intake ( g / day ) on different treatment regimes prescribed large neutral amino acids intake ( g / day ) on different treatment regimes several assumptions underlie the calculated values . first , the dose of amino acid mixture in conventional treatment is set at 1.0  g kg day rather than the rda ( 0.8  g kg day ) , correcting for the assumed 20% of the mixture that is not absorbed in the gastrointestinal tract . second , in conventional treatment , lnaa intake resulting from natural protein intake is not taken into account , as the natural protein in this treatment is assumed to be of relatively low nutritional value . however , the distinction between protein of relatively low and high nutritional value has not been applied to the prescribed lnaa intake at rda of healthy controls , resulting in some inconsistencies . 1 demonstrates that lnaa intake in pku is higher than in healthy controls , regardless of the specific treatment given . moreover , lnaa intake on lnaa supplementation is higher than lnaa intake on conventional treatment . however , the difference in lnaa intake between these two treatment forms is small relative to the difference between conventional treatment and the dietary regimen studied by matalon et al . clearly results in a higher lnaa intake than either conventional therapy or lnaa supplementation , and markedly exceeds lnaa intake of a healthy control . when lnaa treatment is discussed , not only the dose but also the composition is important . it is remarkable that lnaa treatment would need the addition of the non - lnaa lysine and arginine . when the primary aim is to block the entrance of phe into brain , this would favor lnaa supplements consisting of specific amino acids , such as leucine , isoleucine , and methionine , that are considered to be highly effective at decreasing the cerebral phe concentration ( hargreaves and pardridge 1998 ) . following that hypothesis , giving 0.5  g kg day of all lnaa together with lysine and arginine might be changed into 0.5  g kg day of leucine , isoleucine , and/or methionine ( hargreaves and pardridge 1998 ) . when the primary aim is to increase the influx of tyrosine and tryptophan , this would favor the supplementation of only tyrosine and tryptophan to increase the synthesis of the neurotransmitters dopamine and serotonin in the brain . following that hypothesis , giving 0.5  g kg day of all lnaa ( with or without lysine and/or arginine ) might be changed into 0.5  g kg day of tyrosine and tryptophan . this is clearly above the doses ever given to pku patients so far ( lou et al . 1985 , 1987 ; gttler and lou 1986 ; lykkelund et al . 1988 ) . when , however , increasing the cerebral concentrations of all essential amino acids is the primary aim , all essential amino acids should be given in such a way that normal concentrations of all essential amino acids are achieved , taking the km of each amino acid into account . lnaa supplementation may have several treatment aims : reducing brain phe concentrations , reducing plasma phe concentrations , increasing cerebral neurotransmitter concentrations , and increasing cerebral essential amino acid concentrations . although lnaa treatment in pku was initially intended to be unrestricted natural protein intake combined with lnaa supplementation , it should be noted that not all authors studied lnaa supplementation in this manner . first , lnaa supplementation has been found to reduce brain phe concentrations in patients , even at mean plasma phe concentrations > 1,000  mol / l ( pietz et al . second , lnaa supplementation may reduce plasma phe concentrations ( matalon et al . 2006 , 2007 ; sanjurjo et al . 2003 ) , although not all authors report this effect ( pietz et al . 1999 ; koch et al . third , data suggest that supplementation with only tyrosine and tryptophan does not sufficiently correct biochemical and neuropsychological abnormalities . however , recent lnaa trials supplement lnaa at 0.5  g kg day , a dose much higher than that studied for tyrosine and tryptophan supplementation . at this dose , fourth , lnaa supplementation combined with unrestricted natural protein intake has been suggested to increase cerebral essential amino acid concentrations . although usually considered to refer to a single specific treatment modality , this paper shows that lnaa treatment may refer to at least two different lnaa treatment strategies , i.e. , natural protein intake at rda with lnaa supplementation , and conventional dietary therapy combined with lnaa supplementation . in addition , lnaa treatment may refer to supplementation of single amino acids , such as tyrosine , tryptophan , and threonine . these differences in treatment strategies are based on clearly different theories regarding mechanism of action . consequently , more in - depth studies are necessary to investigate the potential role , dose , and composition of lnaa in pku treatment .
notwithstanding the success of the traditional dietary phenylalanine restriction treatment in phenylketonuria ( pku ) , the use of large neutral amino acid ( lnaa ) supplementation rather than phenylalanine restriction has been suggested . 
 this treatment modality deserves attention as it might improve cognitive outcome and quality of life in patients with pku . 
 following various theories about the pathogenesis of cognitive dysfunction in pku , lnaa supplementation may have multiple treatment targets : a specific reduction in brain phenylalanine concentrations , a reduction in blood ( and consequently brain ) phenylalanine concentrations , an increase in brain neurotransmitter concentrations , and an increase in brain essential amino acid concentrations . 
 these treatment targets imply different treatment regimes . 
 this review summarizes the treatment targets and the treatment regimens of lnaa supplementation and discusses the differences in lnaa intake between the classical dietary phenylalanine - restricted diet and several lnaa treatment forms .