oxidative stress in phenylketonuria: future directions

REVIEW

Oxidative stress in Phenylketonuria: future directions

Júlio César Rocha & Maria João Martins

Received: 15 June 2011 /Revised: 24 October 2011 /Accepted: 28 October 2011 /Published online: 25 November 2011# SSIEM and Springer 2011

Abstract Phenylketonuria represents the most prevalentinborn error of amino acid metabolism. In early diagnosedpatients adequate and continued dietary treatment resultsin a good neurologic outcome. Natural protein andphenylalanine-restricted diet, even if rich in fruits andvegetables, represents a serious risk for nutritionaldeficiencies, albeit universally accepted. In the last fewyears, a growing number of reports have been describingoxidative stress as a concern in phenylketonuric patients.The diet itself includes good sources of dietary antiox-idants (phytochemicals, some vitamins and minerals) butalso a risk factor for some deficiencies (selenium, zinc,ubiquinone-10 and L-carnitine). Additionally, the extremestringency of the diet may impose a reduced synthesis ofendogenous antioxidants (like ubiquinone-10 and gluta-thione). Furthermore, increased phenylalanine levels, andits metabolites, may enhance the endogenous synthesis ofreactive species and free radicals and/or interfere with theendogenous synthesis of enzymatic antioxidants (likeglutathione peroxidase). Therefore, oxidative stress willprobably increase, mainly in late diagnosed patients or inthose with bad metabolic control. Considering the knownassociation between oxidative stress, obesity and cardio-vascular disease, it seems advisable to look further to the

impact of oxidative stress on body macromolecules andstructures (like lipoprotein oxidation), especially in phenyl-ketonuric patients with late diagnosis or bad metaboliccontrol, in order to prevent future increased risks. Recom-mendations for PKU patient’s clinical follow-up improve-ment and educational goals are included.

AbbreviationsCAT CatalaseGSH-px Glutathione peroxidaseHDL High density lipoproteinLDL Low density lipoproteinPhe L-PhenylalaninePKU PhenylketonuriaPON ParaoxonasePON1 Paraoxonase 1Q10 Ubiquinone-10; coenzyme Q10RNS Reactive nitrogen speciesROS Reactive oxygen speciesRS Reactive speciesSe SeleniumSOD Superoxide dismutaseTBARS Thiobarbituric acid-reactive speciesZn Zinc

Introduction

There is universal agreement about the success of earlyintroduction of dietary treatment on the prevention ofsevere mental retardation in patients with phenylketonuria(PKU; MIM ID # 261600) (Scriver and Kaufman 2001; vanSpronsen and Enns 2010). Although the benefits of dietarytreatment are undeniable, some discussion still remains

Communicated by: K. Michael Gibson

J. C. Rocha (*)Centro de Genética Médica Jacinto de Magalhães – INSA, IP,Praça Pedro Nunes, 88,4099-028 Porto, Portugale-mail: [email protected]

M. J. MartinsDepartment of Biochemistry (U38-FCT), Faculty of Medicine,University of Porto,4200-319 Porto, Portugal

J Inherit Metab Dis (2012) 35:381–398DOI 10.1007/s10545-011-9417-2

regarding the possible reasons why neuropsychologicalfunctions are not completely normal in some earlydiagnosed and adequately treated patients [with a phenyl-alanine (Phe) restrictive diet, supplemented with Phe-freeprotein substitutes and fortified with macro and micro-nutrients] (de Groot et al. 2010; Enns et al. 2010; Moyle etal. 2007; van Spronsen et al. 2009). Natural proteinrestriction, together with a special synthetic diet, mayincrease the risk of nutritional deficiencies (Enns et al.2010; Feillet and Agostoni 2010; Macdonald et al. 2011).Some of the nutritional deficiencies may result in a lowtotal antioxidant status that can predispose and/or contributeto oxidative stress (Sanayama et al. 2011; Sitta et al. 2006,2009b; van Bakel et al. 2000), though a disease causingeffect cannot be completely excluded (Sitta et al. 2009a).Recently, it was hypothesized that the administration ofappropriate antioxidants as adjuvant agents, in addition tothe usual dietary treatment and supplementation, mayprevent neurological damage in PKU patients (Ribas et al.2011). Considering the interplay between oxidative stressand conditions like obesity and cardiovascular diseases(Ando and Fujita 2009; Farbstein et al. 2010; Furukawa etal. 2004; Hansel et al. 2006; Holvoet et al. 2008a, b), thisreview aims to establish a new perspective for oxidativestress surveillance in PKU patients translated into futuredirections for clinical follow-up and educational goals, inorder to optimize health status during their life-span.

Risk factors for oxidative stress in PKU

During normal metabolism, our cells generate unstable andpotentially harmful substances, designated reactive oxygenspecies [ROS, like hydrogen peroxide, superoxide ion andhydroxyl radical] and reactive nitrogen species (RNS; likenitric oxide and peroxinitrite). ROS and RNS are wellrecognized as playing a dual role as both deleterious andbeneficial species. This delicate balance is a very importantaspect of living organisms and is dependent on a strictregulation. Harmful effects, either mediated by ROS orRNS, result from increased reactive species (RS) concen-tration occurring together with a reduction of enzymaticand non-enzymatic antioxidant defences (Halliwell andGutteridge 2007; Martindale and Holbrook 2002; McCord2000; Seifried et al. 2007; Valko et al. 2006, 2007). Thecontinuous and uncontrolled production of RS results inincreased damage to cellular structures and molecules likeproteins, lipids, carbohydrates and DNA (Bergamini et al.2004; Halliwell and Gutteridge 2007; Seifried et al. 2007;Valko et al. 2006, 2007). The importance of antioxidants inour metabolism and their influence on health and diseasehas been recognized during the last decades (Farbstein et al.2010; Furukawa et al. 2004; Valko et al. 2007; Willett and

MacMahon 1984a, b; Young and Woodside 2001). Theydecrease the chance of an uncontrolled rising of cellular RSconcentrations and prevent damage of cellular structures(Seifried et al. 2007). Cellular antioxidants can be eitherenzymatic or non-enzymatic. The first group includes,among others, enzymes like catalase (CAT), glutathioneperoxidase (GSH-px) and superoxide dismutase (SOD)(Halliwell and Gutteridge 2007; Seifried et al. 2007; Valkoet al. 2007). While SOD has the main function of removingsuperoxide ions, CAT and GSH-px are crucial to combathydrogen peroxide and organic peroxides (Halliwell andGutteridge 2007; Seifried et al. 2007). The non-enzymaticantioxidants include glutathione (major thiol antioxidantand redox buffer of the cell), metal ions sequestrationproteins, thiols, some vitamins (mainly C and E), minerals,carotenoids and phytochemicals, like isoflavones andflavonols (Crozier et al. 2009; Halliwell and Gutteridge2007; Seifried et al. 2007; Valko et al. 2007). Foods likefruits and vegetables, whole grains, legumes, green tea, redwine and vegetable oils are the major sources of dietaryantioxidants (Lindsay and Astley 2002). Thus, diet canseriously modulate antioxidant defences by directly in-creasing the amount of antioxidants or indirectly promotingendogenous antioxidant capacity (Bullon et al. 2009;Haldar et al. 2007). Although positive benefits are welldescribed, it is important to refer that uncontrolled dietarysupplementation of antioxidants, namely vitamins C and E,β-carotene and Se, can also result in negative effects(Halliwell and Gutteridge 2007; Herbert 1994; Martindaleand Holbrook 2002).

In general, the risk of oxidative stress in PKU may berelated with two main aspects: dietary restriction, whichcan alter enzymatic and non-enzymatic antioxidant defen-ces, and excessive production of RS, due to the diseaseitself or to the associated metabolites (Fig. 1).

Dietary restriction

Despite the large spectrum of Phe tolerance, foods likemeats, fish, eggs, standard bread, nuts, beans and dairyproducts are usually removed from PKU patients diet (Blauet al. 2010; MacDonald et al. 2003). PKU diet is not equalto the vegetarian diet, mainly due to the inclusion of proteinsubstitutes, but it has some similarities. Vegetarianism,although usually referred as healthy, can be nutritionally“dangerous” since diet management is necessarily chal-lenging in order to prevent nutrient deficiencies like iron,zinc (Zn), calcium, vitamin D, riboflavin, vitamin B12,vitamin A, n-3 fatty acids and iodine (Craig and Mangels2009; Key et al. 2006). Consequently, some nutritionaldeficiencies are possible in PKU patients (Feillet andAgostoni 2010; Robinson et al. 2000). Additionally,competition during digestive and absorptive processes can

382 J Inherit Metab Dis (2012) 35:381–398

alter the normal bioavailability of some nutrients (Gropperet al. 1993; Lonnerdal 1997; Rocha and Martel 2009).Finally, poor diet compliance in PKU patients can be anextra risk factor for nutritional deficiencies, mainly inprotein and micronutrients, especially after adolescence(Walter and White 2004; Walter et al. 2002).

Altered redox status can result from selenium (Se)(Barretto et al. 2008; Sitta et al. 2011), ubiquinone-10(Q10) (Artuch et al. 1999, 2001, 2004; Colomé et al. 2001,2002), Zn (Barretto et al. 2008) or L-carnitine deficiencies(Schulpis et al. 1990; Sitta et al. 2009b, 2011). Historically,Se deficiency is one important micronutrient deficiencyreported in PKU, although nowadays it is not so frequent(Barretto et al. 2008; Sitta et al. 2011). Reduced Se status(Barretto et al. 2008; Sitta et al. 2011) may impair normalplasma/erythrocyte GSH-px activity (Halliwell andGutteridge 2007; Lu and Holmgren 2009). As a conse-quence, Se deficiency, reducing the ability to cope withthe usual (or increased) production of RS, may result inoxidative stress (van Bakel et al. 2000). Q10 functions asan electron carrier in the mitochondrial electron transportchain and its deficiency may be related with suboptimalfunction of the respiratory chain that associates with ahigher risk for oxidative stress. The reduced form of Q10(Q10H2, ubiquinol) serves an important lipophilic anti-oxidant role by protecting cellular membranes and plasmalipoproteins against free radical induced oxidation as wellas by reducing the oxidized forms of natural antioxidantcompounds (Colomé et al. 2003; Hargreaves 2007). Zn isan inhibitor of the NADPH oxidases, which producesuperoxide ions, as well as a cofactor of SOD thatcatalyzes the dismutation of superoxide ions (Prasad etal. 2004). Zn supplementation has been shown to produce

positive results in several diseases by decreasing thegeneration of inflammatory cytokines and oxidative stress(Prasad 2009), with hypothetical protective effects inatherosclerosis due to its anti-inflammatory and antioxi-dant functions (Bao et al. 2010). Moderate Zn deficiencyin rats enhances lipoprotein oxidation in vitro underliningthe possibility that Zn may affect radical-mediateddamage in vivo, since lipoprotein oxidation in vitroseems to reflect certain oxidative processes in vivo (thatcan contribute to atherosclerosis) (DiSilvestro andBlostein-Fujii 1997; Witztum and Steinberg 2001). L-carnitine also has important antioxidant functions, mainlypreventing peroxidation events while acting as a ROSscavenger (Fritz and Arrigoni-Martelli 1993; Güllçin2006). Additionally, L-carnitine, by mediating fatty acidstransport into mitochondria, may lower fatty acidsavailability for peroxidation (Sitta et al. 2011).

Excessive production of RS

Altered redox status can also result from the increasedamount of free radicals and/or RS caused by the diseaseitself, with a concomitant depletion of the antioxidantdefences (Wajner et al. 2004). Some studies have alreadyhypothesized the possible relationship between oxidativestress and neurological dysfunction in PKU (Ribas et al.2011; Sanayama et al. 2011; Sitta et al. 2011). The in vitroeffect of Phe suggested that oxidative stress is involved inthe physiopathology of PKU (Fernandes et al. 2010).Additionally, the altered antioxidant defences can overcomefree radical and RS generation only up to a certain extent(short-term exposure in early diagnosed patients) after whichthey may become insufficient and not capable anymore to

Oxidative Stress in PKU

The “dietary treatment‘s effect” The “disease’s effect”y

Ph i hibiti“Adjusted” Reduced RS Phe inhibition f?intake of synthesis of production of?antioxidants endogenous caused by the synthesis of?

antioxidants chronic endogenousti id tEx: glutathione exposure to antioxidants E Q10Phe and its Ex:

metabolites

Altered antioxidant status

Adapted ROS/RNSAdapted

??

Visceral (central) obesity / Insulin resistance / Type 2 diabetes / Cardiovascular diseases

Fig. 1 Different contributionsto the oxidative stress in PKUpatients. Phe: L-phenylalanine;Q10: ubiquinone-10,coenzyme Q10; ROS/RNS:reactive oxygen species/reactivenitrogen species;RS: reactive species

J Inherit Metab Dis (2012) 35:381–398 383

cope with free radical and/or RS (in late diagnosed patients)leading to oxidative damage (Sitta et al. 2009a).

Oxidative stress data in PKU patients

PKU diet should be carefully managed in order to preventnutritional deficiencies (Feillet and Agostoni 2010). Takinginto account that daily Se intake positively correlates withserum Se levels, diet compliance plays a crucial role inpreventing Se deficiency (Colomé et al. 2003). In line withthis, it is possible to find early diagnosed PKU patients withplasma Se concentrations similar to healthy controls(Artuch et al. 2004). Plasma/serum Se positively correlateswith plasma/erythrocyte GSH-px activity in several PKUstudies (Colomé et al. 2003; Jochum et al. 1997; Lombecket al. 1982; Sitta et al. 2011; van Bakel et al. 2000; Wilke etal. 1992). However, the study by Sierra and co-workers(Sierra et al. 1998) showed that, in well-controlled PKUpatients, erythrocyte GSH-px activity is lower than incontrols, independently of plasma Se levels (which were inthe normal range) as well as of patients’ blood Phe levels.A positive correlation between GSH-px activity and plasmaSe is present in controls. Thus, GSH-px activity reductiondoes not seem to be caused by Se levels, at least when theyare normal. Besides Se, GSH-px activity in PKU patientsmight be influenced by other factors, such as an unbalancedblood amino acid profile that results in moderately lowlevels of erythrocyte methionine, which, in turn, influencethe correct synthesis of GSH-Px (Butler et al. 1989;Pardridge 1977; Sierra et al. 1998). Erythrocyte GSH-pxactivity is found to be similar (Colomé et al. 2003) orreduced (Sitta et al. 2006) in PKU patients versus controls.Sitta and co-workers (Sitta et al. 2006) also reported thaterythrocyte GSH-px activity is not related to plasma Phelevels. Interestingly, at the time of PKU diagnosis,erythrocyte GSH-px is already low compared to controls(Sirtori et al. 2005). Taken that diet is not yet implementedat the time of diagnosis, this reduction cannot be caused bynutritional deficiencies, mainly in Se, which is essential forthis enzyme (Halliwell and Gutteridge 2007; Lu andHolmgren 2009). A later study showed that bad dietcompliance is associated with a low erythrocyte GSH-pxactivity in PKU patients with either early or late diagnosis(Sitta et al. 2009a). More recently, the negative correlationbetween erythrocyte GSH-px activity and serum Phe levelshas been underlined in early and late diagnosed patientswith normal Se values, which suggests that Phe per seinhibits the enzyme activity (Sanayama et al. 2011).

Activity level results for the erythrocyte antioxidantenzymes SOD and CAT in PKU (versus controls) are alsochallenging: not all three antioxidant enzymes are affectedin all studies or present a similar pattern of variation, when

present. SOD activity has been reported to be low (Sitta etal. 2011; van Bakel et al. 2000) or normal (Artuch et al.2004; Sierra et al. 1998; Sirtori et al. 2005; Sitta et al.2006). On the other hand, metabolic control does not seemto affect CAT activity, that has been found to be low(Artuch et al. 2004). Yet, CAT activity has also been foundunaltered (Sierra et al. 1998; Sirtori et al. 2005; Sitta et al.2006, 2011) and an increase in CAT and SOD activities hasalso been described (Sanayama et al. 2011).

Regarding Q10, low plasma/serum levels have beenfound in PKU patients (Artuch et al. 1999, 2001, 2004;Sanayama et al. 2011) versus controls. Q10 concentrationsseem to deteriorate with age, both in PKU patients andcontrols (Artuch et al. 1999). Therefore, it is not surprisingthe negative correlation present between plasma/serum Q10and plasma Phe concentrations (Artuch et al. 2001, 2004).Low Q10 levels can result from severe animal proteinrestriction, tyrosine deficiency and down regulation of themevalonate pathway (Castillo et al. 1988, 1991a, b;Hargreaves 2007; Weber et al. 1997a, b). Blood Pheconcentrations around 250 μM, which are often seen inPKU patients, seem to inhibit the key enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (cholesterol synthesis) andmevalonate-5-pyrophosphate decarboxylase (mevalonatepathway) (Castillo et al. 1988, 1991a, b; Hargreaves 2007;Sierra et al. 1998). Accordingly, plasma cholesterol isreduced in PKU patients versus controls (Artuch et al.1999) and a positive correlation between serum total Q10and cholesterol values has been found in PKU patients andcontrols (Artuch et al. 1999, 2001). Probably, the mainpotential causative factor for that pattern is increased Phelevels (Artuch et al. 2001). Nevertheless, a recent studyunderlined the tendency for low Q10 levels in PKUpatients, yet without a correlation with blood Phe levels(Sanayama et al. 2011).

Considering the dietary influence on serum/plasma Q10levels, Q10 cellular content has been measured. In wellcontrolled PKU patients total Q10 lymphocyte concentra-tions are lower than in controls, the deficiency beingnegatively associated with plasma Phe levels (Colomé etal. 2002). However, these results were not confirmed in agroup of older (treated and untreated) patients (Hargreaveset al. 2002): mononuclear cell Q10 concentration andmitochondrial respiratory chain complexes II+III activityare not influenced by dietary restriction or increased plasmaPhe levels (Hargreaves et al. 2002). Again, no correlationbetween plasma/blood Phe levels and mononuclear cellQ10 concentrations is found in PKU patients (mainly non-compliant) (Kyprianou et al. 2009). The study does notshow any correlation between mononuclear cell Q10 levelsand tremor (Kyprianou et al. 2009), contradicting aprevious report where Q10 levels are low in patients withtremor (Campistol et al. 2006).


Low plasma total L-carnitine levels are found in PKUpatients, with a positive correlation with plasma Phe levels(Sitta et al. 2009b). The same group showed, after 6 monthsof L-carnitine and Se supplementation, a reversion ofplasma thiobarbituric acid-reactive species (TBARS, mark-er of lipid oxidative damage) and protein sulfhydryloxidation, together with an increase in erythrocyte GSH-px activity (Sitta et al. 2011). Besides Se, other micro-nutrients have an important role on body antioxidantsystems. Although fruits and vegetables are consideredalmost free in the PKU diet (MacDonald et al. 2003),vitamin C intake and blood levels are not different fromthose found in controls (Schulpis et al. 2003). On the otherhand, dietary intake and blood levels of β-carotene and α-tocopherol are higher in PKU patients on-diet (Schulpis etal. 2003). Accordingly, reduced plasma β-carotene in PKUpatients has been reported to negatively correlate withserum Phe levels (Sanayama et al. 2011). The adherence tothe PKU diet is usually related to a higher total plasmaantioxidant status and a reduced lipid ingestion withincreased proportion of polyunsaturated fat (Schulpis et al.2003, 2010). In other words, it is important to advise PKUpatients that diet may offer other advantages besidesmanagement of Phe levels (Schulpis et al. 2003, Schulpiset al. 2005, Schulpis et al. 2007).

Early diagnosed PKU patients on a relaxed diet havereduced plasma total antioxidant status and increased serum8-hydroxy-2-deoxyguanosine (DNA oxidation marker)levels versus patients on-diet and controls. Furthermore,an inverse correlation between total antioxidant status and8-hydroxy-2-deoxyguanosine levels is present in the threegroups (Schulpis et al. 2005). More recently, it has beenshown that damage on DNA from peripheral bloodleucocytes is increased in PKU, most particularly in non-compliant patients (Sitta et al. 2009c). Although a directeffect of Phe on DNA could not be demonstrated, theauthors suggested that it could be a result of oxidative stressmediated by Phe metabolites (Sitta et al. 2009c). PlasmaTBARS and serum oxidized low density lipoprotein (LDL)are increased while plasma total antioxidant reactivity isdecreased in PKU patients (all parameters correlate withserum Phe levels; the latter in a negative way) (Sanayama etal. 2011). A previous study underlined that increasedplasma TBARS and reduced plasma total antioxidantreactivity are already present at the time of diagnosis,which may contribute to the physiopathology of the disease(Sirtori et al. 2005). In the same line, other studies showedthat PKU children have reduced plasma total antioxidantreactivity (Sitta et al. 2009b) and increased plasma TBARS,without any correlation with diet adherence (Sitta et al.2006, 2009b). However, in the group with good compliance,patients with higher plasma total L-carnitine show lowerplasma TBARS and higher plasma total antioxidant reactivity

(Sitta et al. 2009b). Considering the antioxidant properties ofL-carnitine, in good compliant patients, lipid peroxidationprocess and reduced antioxidant defences can be a result, atleast in part, of a low L-carnitine status (Sitta et al. 2009b,2011). Nonetheless, lipid peroxidation also happens in non-compliant patients showing that oxidative stress is really acomplex phenomena (Sitta et al. 2009b).

Another important variable in PKU is the timing ofdiagnosis. Recent results show that plasma protein and lipidoxidative damage is increased in late diagnosed patients(Sitta et al. 2009a). The same study reports a reducedantioxidant status in both early and late diagnosed patients(Sitta et al. 2009a). Oxidative damage markers are onlyevident in late diagnosed patients, which may be related tothe deleterious effects of the long term exposure toextremely high Phe levels, besides a possible depletion ofaltered antioxidant defences (Sitta et al. 2009a). Thus,caution is needed when trying to interpret antioxidant statusin PKU, since short and long-term effects of Phe exposuremay not entail the same outcome for patients. Nevertheless,taking into consideration all published data, it seemsprudent to inform patients with late diagnosis, who do notwant to be treated, that they probably will be at a higherrisk of oxidative stress and related morbidity.

Analysing Table 1, it can be established that a) studydesigns have important differences in terms of the follow-up time-period, patient’s classification and good and badmetabolic control definitions and b) detailed characteriza-tion of free dietary habits is missing. Another aspect thatshould be taken into account is the full nutritionalcomposition of the Phe-free protein substitutes that wereused in those studies [most particularly in what concernsthe antioxidant components, point recently highlighted bySitta and co-workers (Sitta et al. 2011)] since theircomposition has been substantially modified in the lastfew years with the introduction of various macro andmicronutrients, like tyrosine, essential fatty acids, L-carnitine, Se and Zn. Altogether, care is needed whentrying to generalize and/or compare data obtained fromdistinct studies, most particularly the older ones.

Recommendations for PKU patient’s clinical follow-upimprovement and educational goals

The association between blood lipids and risk of cardio-vascular diseases is well known. Besides their blood levels,their structures and functions deserve special attention.High density lipoprotein (HDL) and LDL are two importantexamples. HDL has important antioxidant, anti-inflammatory,antithrombotic, antiatherosclerotic and vasodilatory proper-ties, in addition to its role on the reverse cholesterol transport,which can be related to the presence of the paraoxonase 1


Tab

le1

Sum

maryof

therepo

rted

human

stud

ieswith

focuson

oxidativestress

inPKU;stud

iesarepresentedaccordingto

publishing

date,andalph

abetical

orderin

each

year

Firstauthor

anddate

/coun

try/journal

Patientscharacterizatio

n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

(Jochu

met

al.19

97)

87classicalPKU

patientsun

derdietarytreatm

ent,

5–15

y(average

age9.5y)

Toinvestigate,

during

routineclinical

visits,plasma

andwho

lebloo

dSelevelsas

wellas

plasmaand

erythrocyteGSH-Pxactiv

ities

inPKU

patients;to

evaluate

Phe

levels.

-Sestatus

comprom

ised

inPKUchild

ren(allp<0.00

1vs

controls).

Germany

Investigationdu

ring

routineclinical

visits;no

inform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

n

-Plasm

aanderythrocyteGSH-PxSe-depend

ent

activ

ities

lower

inPKU

vscontrols(bothvalues

p<0.00

1).

ActaPaediatrica

Noinform

ationprov

ided

regardingdiet

orPhe

levels

-Plasm

aandwho

lebloo

dSecorrelated

with

each

otherandwith

plasmaanderythrocyteGSH-Px.

-Plasm

aandwho

lebloo

dSelevelscorrelated

with

the

meanPhe

values

(plasm

aSe,r=

0.40

88;p<0.00

1)of

thePKU

child

ren.

(Sierraet

al.19

98)

42well-controlledPKU

patients,1m–1

7y

(average

age7.12

y),initially

120–56

0μmol/L

Phe

Tomeasure

erythrocyteantio

xidant

enzymes

activ

ities

(SOD,GSH-px,

GRandCAT)andα-tocop

herol,

andplasmaSein

PKU

andm-H

PApatients;to

evaluate

bloo

dPhe

levels.

-Nodifferencesin

erythrocyteα-tocop

herollevels

amon

gthethreegrou

ps.

Spain

28m-H

PApatients,with

free

diet,1m–8

y(average

age3.2y),initially

120–

360μmol/L

Phe

Indexof

dietarycontrol:half-yearmedians

andthe

meanof

allthesemedians

(calculatedby

data

redu

ction).

-Erythrocyte

GSH-pxactiv

ities

lower

(p<0.00

1)in

both

PKU

andm-H

PApatientsvs

controlgrou

p,with

outdifferencesbetweenthetwopatientsgrou

ps.

Clin

icaChimicaActa

Noinform

ationprov

ided

regardingdiet

ordu

ratio

nof

patientsmon

itorizatio

n-Nodifferencesin

erythrocyteSOD,GRandCAT

activ

ities

amon

gallgrou

ps.

45controls,1m–1

7y(average

age6.5y)

-Positive

correlationbetweenerythrocyteGSH-Px

activ

ityandplasmaSelevelsin

controlgrou

p(r=0.56

22;p=0.01

5)bu

tno

neforpatients[alsono

correlationregardingerythrocyteGSH-Pxandbloo

dPhe

forpatients(orindexof

dietarycontrolforPKU

grou

p)].

-Plasm

aSelevelsin

theno

rmal

rang

ein

both

grou

psof

patients;no

differencesbetweenpatientsgrou

psin

either

bloo

dPhe

orplasmaSelevels.

(Artuchet

al.19

99)

43PKU

patients,1m–3

3y(m

edianage13

y),

Phe

results:59–1

044μmol/L

(median37

5μmol/L)

Toevaluate

(retrospectiv

elyandtransversally

)serum

totalub

iquino

ne-10(oxidized+redu

ced)

concen-

trations

andplasmaa

cholesterol,Tyr

andPhe

levels

inPKU

patients;to

evaluate

Tyr

daily

intake.

-Serum

totalub

iquino

ne-10concentrations

lower

inPKU

vscontrols(p<0.01

forpatientsaged

1m

to<8y,

p<0.00

005for8–33

y).

Spain

Diet:supp

lementedwith

aTyr-enrichedam

inoacid

mixture

-Higherprevalence

oflow

serum

totalub

iquino

ne-10

inolderPKU

patientsthan

inyo

ungerpatients(43%

vs28

%,respectiv

ely).

The

American

Journal

ofClin

ical

Nutritio

nNoinform

ationprov

ided

regardingdu

ratio

nof

patients

mon

itorizatio

n-Negativecorrelations

betweenserum

total

ubiquino

ne-10concentrations

andagein

patients

(r=−0

.36;

p<0.05

)andcontrols(r=−0

.383

;p<0.00

1).

102controlsfrom

referenceage-matched

popu

latio

n-Plasm

acholesteroldecreasedin

PKU

vscontrols

(p<0.01

forpatientsaged

<14

y,p<0.00

005for

14–33y).


Tab

le1

(con

tinued)

Firstauthor

anddate

/coun

try/journal


n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

-Positive

correlationbetweenserum

totalub

iquino

ne-

10andplasmacholesterolv

aluesin

patients(r=0.503;

p<0.001)

andcontrols(r=0.485;

p<0.00001).

-Nocorrelations

betweenplasmaTyr

andserum

total

ubiquino

ne-10levels,Tyr

daily

intake

andserum

totalub

iquino

ne-10concentrations,or

plasmaPhe

andserum

totalub

iquino

ne-10concentrations

ineither

grou

p.

(Fisberg

etal.19

99)

42PKU

patients,1–

12y

Tomeasure

plasmaZnandCulevelsas

wellas

CuZ

nSOD

erythrocyteactiv

ityin

PKU

patients.

-Plasm

aZnconcentrations

ofPKUchild

renolderthan

7ylower

than

incontrols(p=0.05

),bu

tstill

with

intheno

rmal

rang

e;in

child

renyo

ungerthan

7yno

substantialdifferencesfoun

dbetweenthetwo

grou

ps.

Brazil

Twogrou

ps:<7y(n=24

)and≥7y(n=18

)-Plasm

aCuconcentrations

andCuZ

nSODerythrocyte

activ

ities

similarin

PKU

child

renandcontrols.

Nutritio

nDiet:Phe-freeplus

vitamin,mineral

saltand

traceelem

entsupp

lement

Noinform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

nor

Phe

levelsof

PKU

patients

31controls,<7yn=(9)and≥7y(n=22

)

(van

Bakel

etal.20

00)

24PKU

patients,4–

17y(m

eanage9.65

±4.06

y)Toinvestigatetheeffectsof

low

plasmaSelevels

onplasmaTA

S,uricacid,album

inandα-tocop

herol

aswellas

onerythrocyteantio

xidant

system

(glutathione

peroxidase,Cu/ZnS

OD,glutathion

eredu

ctaseandglutathion

etransferaseactiv

ities

andGSH

content)in

PKU

patients.

-Plasm

aSevalues

lower

inPKU

patientsvs

HPA

patients(p<0.01

).

Switzerland

10HPA

patients,4–18

y(m

eanage9.08

±5.17

y)-Plasm

aSevalues

lower

inPKU

andHPA

patientsvs

controls(p<0.00

1andp<0.01

,respectiv

ely).

The

American

Journal

ofClin

ical

Nutritio

nBlood

samples

werecollected

4to

6tim

esov

era

period

of14

m-Plasm

aTA

Slower

inbo

thgrou

psof

patientsvs

controls(p<0.01

);patient

grou

psdidno

tdiffer

from

each

other.In

patients,on

lyplasmauric

acid

values

correlated

with

plasmaSevalues

(r=0.37

;p<0.05

).Nodifferencesforplasmauric

acid,albu

min

andα-

tocoph

erol

amon

gthethreegrou

ps.

Noinform

ationprov

ided

regardingdiet

orPhe

levels

ofPKU

orHPA

patients

-In

patients,plasmaSecorrelated

with

erythrocyte

glutathion

eperoxidase

activ

ity(r=0.76

;p<0.00

0001

)andGSH

(r=0.40

;p<0.05

).

42controls,1–19

y(m

eanage11.18±4.84

y)-Low

ererythrocyteglutathion

eperoxidase

activ

ityin

PKU

patientsvs

HPA

patientsandcontrols(p<0.05

andp<0.00

1,respectiv

ely).

-Erythrocyte

GSH

values

lower

inPKU

patientsthan

incontrols(p<0.05

).

-ErythrocyteCu/ZnS

ODactiv

itylower

inbo

thgrou

psof

patientsvs

controls(p<0.05

);patient

grou

psdid

notdiffer

from

each

other.


Tab

le1

(con

tinued)

Firstauthor

anddate

/coun

try/journal


n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

-Nodifferencesforerythrocyteglutathion

eredu

ctase

andglutathion

etransferaseactiv

ities

amon

gthethree

grou

ps.

(Artuchet

al.20

01)

30PKU

patientsun

derdiet

(with

good

metabolic

control),5m–3

5y(m

edianage7y),initially

205–

643μmol/L

Phe

(median34

1μmol/L)

Toinvestigateexistin

gcorrelations

betweenplasma

low

Tyr

orhigh

Phe

levelsor

treatm

entwith

Phe-

restricted

diet

anddecreasedserum

totalub

iquino

ne-

10concentrations

inPKU

patients;to

calculatedaily

cholesterolintake

andmeasure

serum

cholesterol.

-Low

serum

totalub

iquino

ne-10values

foun

din

40%

ofthepatients.

Spain

Diet:Phe-restricteddiet

supp

lementedwith

aTyr-enrichedam

inoacid

mixture

Indexof

dietarycontrol:averageof

themedians

ofplasmaPhe

concentrations

obtained

every6m

inthe

metabolic

controlof

patients.

-Plasm

aTyr

values

lower

inPKU

patients(p<0.01

)than

incontrols.

Journalof

Inherited

Metabolic

Disease

2yof

patientsmon

itorizatio

n-Negativecorrelations

betweenserum

total

ubiquino

ne-10andplasmaPhe

levels(r=−0

.44;

p<0.05

)andindexof

dietarycontrol(r=−0

.46;

p<0.01

)in

PKU

patients.

102controls,1m–3

5y

-Positive

correlations

betweenserum

total

ubiquino

ne-10andcholesterolconcentrations

(r=0.39

;p<0.05

)andbetweenageandindexof

dietarycontrol(r=0.7;

p<0.00

01).Nocorrelations

either

betweenplasmaTyr

andserum

total

ubiquino

ne-10values

orbetweendaily

cholesterol

intake

andserum

cholesterolor

serum

total

ubiquino

ne-10levels.

(Colom

éet

al.20

02)

23well-controlledPKU

patients(Phe-restricted

diet

supp

lementedwith

aTyr-enrichedam

ino

acid

mixture),8–36

y(m

edianage16

y)

Tocompare

totalQ10

concentrations

inlymph

ocytes

ofPKU

patientsvs

controls;to

evaluate

plasmaPhe

levels.

-Lym

phocytetotalQ10

concentrations

redu

cedin

PKU

patientsvs

controls(p<0.00

1).

Spain

IDC

results:29

5–89

9μmol/L

(median45

6μmol/L)

Indexof

dietarycontrol:medianplasmaPhe

concentrations

ofthelast6mon

ths.

-17

PKU

patientswith

lymph

ocytetotalQ10

concentrations

below

thelower

limitof

thereference

interval

establishedin

controlpo

pulatio

n.

Clin

ical

Biochem

istry

25age-matched

controls

-Lym

phocytetotalQ10

levelsnegativ

elyassociated

with

plasmaPhe

levels(r=−0

.441

;p<0.05

).

(Hargreaveset

al.20

02)

14PKU

patientson

diet,15

–44y(m

eanage30

.2±

9.7y,

restricted

Phe

intake

accordingto

Phe

hydrox

ylasemutationseverity

+otheressential

aminoacids,vitamins,mineralsandtraceelem

ents

asrequ

ired),meanPhe

results:49

6.4±22

0μmol/L

Toevaluate

theeffect

ofdietaryrestrictionand

elevated

plasmaPhe

levelson

bloo

dmon

onuclear

cellCoQ

10levelsandtheactiv

ityof

the

mito

chon

drialrespiratorychaincomplex

II+III

(succinate:cytochrom

e-credu

ctase)

inPKU

patients.

-The

bloo

dmon

onuclear

cellCoQ

10concentrations

notfoun

dto

bedifferentbetweenthecontrol,treated

andun

treatedPKU

patient

grou

ps.

UK

12PKU

patientsoffdiet,18

–46y(m

eanage29

.9±

8.0y,

access

tofullrang

eof

food

stuffs

includ

ing

meat,fish

anddairyprod

uctswith

noexclusions),

meanPhe

results:97

3.1±41

5.3μmol/L

-Mon

onuclear

cellmito

chon

drialcomplex

II+III

activ

ityno

tfoun

dto

bedifferentbetweenthethree

grou

ps.

Journalof

Inherited

Metabolic

Disease

Noinform

ationprov

ided

regardingdu

ratio

nof

patients

mon

itorizatio

n


Tab

le1

(con

tinued)

Firstauthor

anddate

/coun

try/journal


n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

17controls,16

–48y(m

eanage34

.6±9.5y)

(Colom

éet

al.20

03)

58PKU

patientsun

derdietarytreatm

ent,

2–36

y(m

edianage13

y),Phe

results:

152–

1407

μmol/L

(median60

4μmol/L)

Tomeasure

[inacross-sectionalstud

y,with

PKU

patientsstratifiedin

twogrou

ps:grou

p1(low

Q10

levels)andgrou

p2(normal

Q10

levels)]

serum

aQ10

,retin

ol,tocoph

erol

andSelevels,as

wellas

plasmaPhe,MDA

andascorbateconcen-

trations

anderythrocytec

GPX

activ

ity.Toevaluate

Se,

ascorbate,

retin

olandtocoph

erol

daily

ingestion.

-Serum

Q10

levelslower

inglob

alPKU

vsmod

erate

HPA

(p=0.02

5)andcontrols(p<0.00

1);PKU

grou

p1values

lower

(allvalues

p<0.00

1)than

PKU

grou

p2,

mod

erateHPA

andcontrols.

Spain

Diet:Phe-restricteddiet

+-Serum

Selevelslower

inglob

alPKU

andin

PKU

grou

p1vs

mod

erateHPA

(p<0.00

5)andcontrols

(p<0.00

1).

The

American

Journal

ofClin

ical

Nutritio

nTyr-enrichedam

inoacid

mixture

-Serum

tocoph

erol

values

lower

inPKU

grou

p1

vsPKU

grou

p2(p=0.00

5)andmod

erateHPA

(p=0.02

9).

30mod

erateHPA

patientswith

nodiet

restriction,

3–17

y(m

edian7.5y)

-Plasm

aMDA

high

erin

glob

alPKU

vscontrols

(p≤0

.005

);high

erin

PKU

grou

p1vs

PKU

grou

p2

(p=0.04

8),HPA

(p=0.02

5)andcontrols(p≤0

.005

).

Noinform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

n-Nodifferencesbetweenmod

erateHPA

andcontrol

grou

psin

anyof

thevariablesstud

iedbu

tforplasma

Phe

concentrations

(p<0.00

1).Nodifferencesin

erythrocyteGPX

activ

ityor

inplasmaascorbateand

serum

retin

olvalues

betweenallgrou

ps.No

differencesin

daily

intakesof

tocoph

erol,retin

ol,

ascorbateandSeor

plasmaPhe

values

betweenPKU

grou

ps1and2.

Nocorrelations

forplasma/serum

concentrations

ofvitamins,Phe

andMDA

anddaily

intakesof

vitaminsandSein

glob

alPKU.

58controls,1–

38y(m

edianage12

y)-In

glob

alPKU

positiv

ecorrelations

betweenserum

Q10

andtocoph

erol

values

(r=0.51

0;p<0.00

1),

erythrocyteGPXactiv

ityandserum

Seconcentration

(r=0.33

7;p=0.00

7)anddaily

Seintake

andserum

Selevel(r=0.36

4;p=0.03

1).

(Schulpiset

al.20

03)

22PKU

patientson

strict

diet,meanageof

7.7±3.2y;

meanannu

alPhe

levels:29

2±60

μmol/L

Toevaluate

theeffect

ofdiet

onplasmaTA

Sin

PKU

patients.Tomeasure

nutrientsingestion

aswellas

bloo

dlevelsof

lipids,vitamin

C,

β-caroteneandα-tocop

herol.

-Vitamin

Cintake

andbloo

dlevelssimilaram

ong

grou

ps.

Greece

24PKUpatientswith

high

Phe

levels,m

eanageof

8.0

±3.6y;

meanannu

alPhe

levels:89

5±54

μmol/L

-Intake

andbloo

dlevelsof

β-caroteneandα-

tocoph

erol

high

erin

diet

adherentsvs

theothertwo

grou

ps(p<0.00

1).

Europ

eanJournalof

Clin

ical

Nutritio

nDiet:allpatientson

aminoacid

mixture

intake

with

vitamins,traceelem

entsandcarnitine,

butwith

outfat

-Lipid

intakesandtheirbloo

dlevelslower

inpatients

onthestrict

diet.

40controls,meanageof

7.68

±2.6y

-Plasm

aTA

Shigh

erin

diet

adherents.

-Positive

correlations

betweenantio

xidant

vitamin

bloo

dlevelsandplasmaTA

Sin

allgrou

ps,

especially

indiet

adherent

patients.


Tab

le1

(con

tinued)

Firstauthor

anddate

/coun

try/journal


n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

-Antioxidant

status

high

erin

PKU

patientson

strict

diet

vstheothertwogrou

ps(p<0.00

1).

(Artuchet

al.20

04)

46earlydiagno

sedPKU

patients(divided

into

two

grou

psaccordingto

indexof

dietarycontrol),

6m–3

4y(average

12.9

y),un

derdietary

treatm

entandfollo

w-up

Tomeasure,in

along

itudinalstud

y,ov

er3y,

plasmaa

Se,

tocoph

erol

andQ10

levelsas

well

aserythrocyteantio

xidant

enzymes

activ

ities

(CAT,

SOD,GSH-PxandGR)in

PKU

patients;to

evaluate

plasmaPhe

levels.

-Plasm

aQ10

concentrations

lower

inPKU

patientsvs

controls(p<0.00

01);deteriorationwith

patients’

age

(r2=−0

.124

;p=0.01

6)andwith

indexof

dietary

control(r2=−0

.094

;p=0.02

7).Plasm

aQ10

values

decreasedov

erthedu

ratio

nof

thestud

y,in

both

PKU

grou

ps1(p=0.00

4)and2(p=0.04

3).

Spain

Goo

dmetabolic

control:6m–2

2y(average

age

6.5y)

and

initially

ðÞa

verage

IDC¼

279�1

07mM

Indexof

dietarycontrol:averageof

themedians

ofplasmaPhe

concentrations

obtained

every

6mon

thsof

patientsmetabolic

mon

itorizatio

n.

-Plasm

aQ10

concentrations

positiv

elyassociated

with

plasmatocoph

erol

levels(r2=0.22

3;p=0.00

2).

Clin

ical

Biochem

istry

Bad

metabolic

control:11–3

4y(average

age11.2

y)and

initially

ðÞa

verage

IDC¼

734�1

64mM

-Plasm

aSelevelsno

tim

paired

inbo

thgrou

psof

patientsun

derdietarytreatm

ent.

Diet:allpatientson

Phe-restricteddiet

with

aTyr-enrichedam

inoacid

mixture

with

Se

-Erythrocyte

CATactiv

ityredu

ced(p=0.00

1)in

patients,bu

tno

differencesfoun

din

SOD,GSH-Px

andGRactiv

ities

vscontrols.CATactiv

itynega-

tivelyassociated

with

plasmaPhe

values

(r2=

−0.152

;p=0.04

5)on

lyin

thewell-controlledPKU

patients.

58controls,1–

38y(average

age12

y)

(Schulpiset

al.20

05)

24PKU

patientswith

good

diet

compliance,

mean

age7.7±3.2y),initially

meanannu

al29

2±60

μmol/L

Phe

Toevaluate

theeffect

ofplasmaTA

Sandbloo

dPhe

onserum

8-OHdG

levelsin

(early

diagno

sed)

PKU

patients.

-Plasm

aTA

Slower

inpatientson

relaxeddiet

(p<0.00

1vs

patientson

good

diet

andp<0.00

01vs

controls).

Greece

25PKU

patientswith

relaxeddiet

compliance,

meanage8.0±3.6y,

initially

meanannu

al89

5±54

μmol/L

Phe

-Serum

8-OHdG

levelshigh

erin

patientson

relaxed

diet(p<0.00

1vs

patientson

good

dietandp<0.00

01vs

controls).

Clin

ical

Biochem

istry

Diet:Phe-freemixture

ofam

inoacids+vitamins,

traceelem

ents+carnitine,no

fatof

anykind

-TA

Sand8-OHdG

levelssimilarin

patientson

good

diet

andcontrols.

Noinform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

n-InversecorrelationbetweenTA

Sand8-OHdG

levels

inallgrou

ps.

24controls,meanage7.7±2.6y

-Blood

Phe

levelsin

closepo

sitiv

ecorrelationwith

serum

8-OHdG

levelsin

both

grou

psof

PKU

patients.

(Sirtoriet

al.20

05)

20PKU

patients,2–20

yEvaluationof

plasmaTBARSandTA

Rand

erythrocyteantio

xidant

enzymes

activ

ities

(CAT,

SOD

andGSH-px)

inPKU

patients

bysamples

obtained

attim

eof

diagno

sis.

-TBARShigh

erin

theplasmaof

PKU

patientsvs

controls(p<0.05

).

Brazil/Argentin

aPatientsun

derno

proteinor

Phe

dietary

restricted

therapy

-TA

Rredu

cedin

theplasmaof

PKU

patientsvs

controls(p<0.05

).

Biochim

icaet

Bioph

ysicaActa

Inclusioncriteria:

Phe

plasmalevelsat

least60

0μmol/L,meanvalue=

1160

μmol/L

Con

trols,age-matched

individu

als

-Decreaseof

erythrocyteGSH-pxactiv

ityin

PKU

grou

pvs

controls(p<0.05

).

-Nodifferencesin

erythrocyteCATandSOD


Tab

le1

(con

tinued)

Firstauthor

anddate

/coun

try/journal


n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

activ

ities

betweengrou

ps.

(Sittaet

al.20

06)

14PKU

patients,7well-controlled,

8.0±2.89

y(initially

5.2-9.4mg/dL

Phe,average6.57

±1.03

mg/dL

)and7bad-controlled,

9.28

±3.30

y(initially

17.3

-21.1mg/dL

Phe,average

19.58±1.50

mg/dL

)

TocorrelateplasmaPhe

concentrations

with

theextensionof

theox

idativestress

(plasm

aTBARSandTA

R,andactiv

ities

ofthe

erythrocyteantio

xidant

enzymes

CAT,

SOD,

GSH-Px)

inPKU

patients.

-Plasm

aTBARShigh

erin

both

grou

psof

PKU

patientsvs

controls(p<0.01

);no

differencesbetween

thetwogrou

psof

PKU

patients.

Brazil

Noinform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

n-Decreaseof

plasmaTA

RanderythrocyteGSH-Px

activ

ity(allp<0.01

)in

both

grou

psof

patientsvs

controls;no

differencesbetweenthePKU

grou

ps.

Metabolic

Brain

Disease

Diet:allpatientson

naturalproteinrestricted

diet

+Phe-freeam

inoacid

mixture

+vitamins

andminerals

-NocorrelationbetweenplasmaPhe

levelsand

erythrocyteGSH-Pxactiv

ityor

plasmaTBARSor

TARvalues

inthesepatients.

7controls,8.63

±2.26

y-CATandSOD

erythrocyteactiv

ities

wereno

taltered

inPKU

patients.

(Schulpiset

al.20

07)

17po

orly

controlledPKU

child

ren;

meanage

6.8±1.4y

Tocompare

PON1andPON-A

rylactiv

ities,

TAC,lip

idprofile

(including

Apo

AI)and

Phe

levelsin

PKU

patients.

-Phe

differed

amon

gthegrou

ps.

Greece

Offdiet

period

of3mon

ths;meanPhe

levels:

1760

±16

0μmol/L

-Lipidsandlip

oproteins,except

HDL-C

andApo

AI,

high

erwhenoffdiet

than

thoseon

diet.HDL-C

and

Apo

AIsimilarin

patientsandcontrols.

Europ

eanJournalof

Clin

ical

Nutritio

nAfter

therequ

estof

follo

wingdiet

strictly

for

30days:meanPhe

levels:49

2±10

0μmol/L

-TA

Clower

whenpatientswereoffdiet

vswhenthey

adheredto

diet

andcontrols(p<0.00

1).

Diet:daily

proteinintake

largelyreplaced

with

anam

inoacid

mixture

with

vitaminsandtrace

elem

ents,bu

twith

outfat

-PON1andPON-arylactiv

ities

redu

cedin

child

ren

with

high

Phe

vsthosewith

low

bloo

dPhe

levels

andcontrols(p<0.00

1).

24controls

-Enzym

eactiv

ities

positiv

elycorrelated

with

HDL-C

andApo

AIwhenPKU

patientswereon

diet

and

controlsas

wellas

with

TAC

inallthegrou

ps;

negativ

elycorrelated

with

Phe

levels.

(Kyp

rianou

etal.20

09)

39PKU

patients(5

onaPhe-restricteddiet,

34on

ano

rmal

diet),20–4

9y(m

eanage

33.7±7.7y)

Tocompare

plasmaandbloo

d-spot

Phe

and

Tyr,andmon

onuclear

CoQ

10levelsin

PKU

patients(w

ithandwith

outtrem

or).

-Nodifference

inCoQ

10levelsor

thepresence/

absenceof

trem

orin

patientson

aPhe-restricteddiet

vsthoseon

anun

restricted

diet.

UK

Patientson

restricted

diet

sent

inmon

thly

bloo

dspotsforPhe

measurementandallpatients,

whether

orno

ton

restricted

diet,hadplasma

Phe

measuredon

ceor

twiceannu

ally

Toexpo

sehu

man

1321

N1astrocytom

acells

toPhe

(300

or90

0μmol/L,for96

h)and

evaluate

mito

chon

drialrespiratorychain

complex

Iactiv

ityandCoQ

10level.

-Nodifferencesin

Phe,Tyr

ormon

onuclear

CoQ

10levelsin

patientswith

trem

orvs

patientswith

out

trem

or.

Journalof

Inherited

Metabolic

Disease

Noinform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

nor

diet

17(out

of39

;44

%)PKU

patientswith

trem

or

-NocorrelationbetweenplasmaPhe

orTyr

(atthe

timeof

mon

onuclear

CoQ

10measurement),mean

plasmaPhe

orTyr

ormeanbloo

dPhe

and

mon

onuclear

CoQ

10levels.Positive

correlation

betweenmeanbloo

dTyr

levelsandCoQ

10levels

(r2=0.35

6;p=0.01

).


Tab

le1

(con

tinued)

Firstauthor

anddate

/coun

try/journal


n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

Cellcultu

reexpo

sure

tohigh

Phe

levels

-Phe

didno

tredu

cecomplex

Iactiv

ityor

CoQ

10biosyn

thesisin

cellcultu

re.

(Sittaet

al.20

09b)

20classicalPKU

patients

Todeterm

ineplasmatotalL-carnitin

elevelsand

plasmaTBARSandTA

Rin

twogrou

psof

PKU

patients:with

good

orpo

oradherenceto

diet/

treatm

ent;to

evaluate

plasmaPhe

levels.

-Plasm

atotalL-carnitin

elevelslower

inpatientswho

strictly

adheredto

diet

(p<0.01

)vs

controlsand

patientswho

didno

tcomplywith

diet.

Brazil

Group

A(n=10

)with

good

diet

compliance,

mean

age8.28

±2.87

y(initially

meanPhe:39

6.9±

46.8

μmol/L),andgrou

pB(n=10

)with

nostrict

adherenceto

diet,meanage9.59

±3.96

y(initially

meanPhe:10

96.7±78

.3μmol/L)

-Positive

correlationbetweenplasmatotalL-carnitin

eandplasmaPhe

levels(r=0.92

1;p<0.01

).

CellularandMolecular

Neurobiolog

yPatientsmon

itorizatio

nfor7.07

±2.85

y-Plasm

aTBARShigh

er(p<0.01

)andplasmaTA

Rlower

(p<0.01

)in

both

grou

psof

PKU

patientsvs

controls.

Diet:low

proteindiet

+synthetic

aminoacids

form

ula,

noPhe,L-carnitin

eor

Se

-NegativecorrelationbetweenplasmaTBARSvalues

andplasmatotalL-carnitin

econcentrations

(r=−0

.577

;p<0.05

)andpo

sitiv

ecorrelationbetween

plasmaTA

Rvalues

andtotalplasmaL-carnitin

elevels(r=0.60

6;p<0.05

)in

well-treatedpatients.

10age-matched

controlchild

ren:

meanage

9.41

±3.50

y-NocorrelationbetweenplasmaTBARSandTA

Rand

plasmatotalL-carnitin

evalues

inthegrou

pof

patientswith

high

plasmaPhe

levelsandno

rmaltotal

plasmaL-carnitin

elevels(group

B).

(Sittaet

al.20

09a)

20classicalPKU

patientswith

poor

treatm

ent

control,10

with

neon

atal

diagno

sis,8.9±2.1y,

and10

with

late

diagno

sis,9.2±1.7y

Tocompare

theinfluenceof

timeexpo

sitio

nto

high

bloo

dPhe

levelson

erythrocyte(G

SH

leveland

GSH-Pxactiv

ity)andplasma(TAR,T

BARS,p

rotein

carbon

ylsandsulfhy

dryl

contents)ox

idativestress

parametersin

PKU

patientswith

earlyandlate

diagno

sis,who

didno

tadhere

toproteinrestricted

diet.

-Erythrocyte

GSH-Pxactiv

ity(p<0.05

),GSH

content

(p<0.01

)andplasmaTA

R(p<0.01

)lower

inearly

andlate

diagno

sedpatientsvs

controls;similar

values

inPKU

grou

ps.

Brazil

Average

bloo

dPhe

levels(calculatedfrom

the

variou

smeasurementsob

tained

every2m):

551±24

5μmol/L

forgrou

pA

and51

8±25

3μmol/L

forgrou

pB).The

patients’

bloo

dPhe

levelsat

themom

entof

thetestswere87

4±

84.4

μmol/L

forgrou

pA

and83

5±78

.9μmol/L

forgrou

pB

-Plasm

aTBARS(p<0.01

)andproteincarbon

yls

(p<0.01

)high

erin

patientswith

late

diagno

sisvs

patientswith

earlydiagno

sisandcontrols;similar

values

inthesetwolastgrou

ps.

InternationalJournal

ofDevelop

mental

Neuroscience

Noinform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

n-Plasm

aproteinsulfhy

dryls(p<0.01

)lower

inpatients

with

late

diagno

sisvs

patientswith

earlydiagno

sis

andcontrols.

Diet:restricted

proteindiet

+essentialam

ino

acid

synthetic

mixture

(noL-carnitin

eor

Se);

Phe

andTyr

accordingto

patients’

age

10age-matched

controls

(Sittaet

al.20

09c)

18PKU

patients[8

with

good

diet

compliance

(meaninitial

bloo

dPhe:39

6.4±

151.6μmol/L)

and10

with

badcompliance(m

eaninitial

bloo

d

Toanalysein

vitroDNA

damagein

leucocytes

from

controls,by

incubatio

nwith

Phe

(100

,25

0,50

0,10

00or

2500

μmol/L,for6h).

-Positive

dose-dependent

effect

ofPhe

onin

vitro

DNA

damagein

leuk

ocytes

from

norm

alindividu

als

(until[Phe]=

1000

μmol/L;p<0.01

).


Tab

le1

(con

tinued)

Firstauthor

anddate

/coun

try/journal


n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

Phe:84

8.8±

150.8μmol/L)],3–

25y(age

atdiagno

sis25

.4±23

.1m)(patientsdividedinto

twogrou

psaccordingto

theirannu

alaverage

Phe

bloo

dlevels)

Brazil

Patientsmon

itorizatio

nfor11.7±4.7y

Toanalysein

vivo

DNA

damagein

leucocytes

from

treatedPKU

patients;to

evaluate

bloo

db

Phe

levels.

-DNAdamageindexhigh

erin

PKUpatientswith

high

bloo

dPhe

levelsvs

well-treatedpatients(p<0.00

1)andcontrols(p<0.00

01).

-Well-treatedPKUpatientswith

greaterDNA

damage

vscontrols(p<0.00

01).

MutationResearch

Diet:natural-protein-restricted

diet

+Phe-free

aminoacid

mixture

+vitaminsand

minerals(noL-carnitin

eandSe)

17age-matched

controls

(Sanayam

aet

al.20

11)

40PKU

patients,15–5

0y;

meanage28

.4±

11.3

y(initialbloo

dPhe:18

0–18

00μmol/L)

Toevaluate

oxidativelesion

markers

(TBARS,

TAR,arginine

aandcitrullin

eain

plasma;

MDA-LDL,ADMA

andNOxin

serum;

acrolein-lysineand8-OHdG

inurine).

-Plasm

aTBARS,serum

MDA-LDLandurinary

acrolein–lysinelevelshigh

erin

PKU

patientsvs

controls;while

urinary8-OHdG

levelssimilarin

the

twogrou

psandplasmaTA

Rlower

inPKU

patients

vscontrols.

Japan

Patients<

33y:

neon

atal

diagno

sis;

Patients>

34y:

diagno

sedbetween1–6y

Toevaluate

CoQ

10,β-caroteneandα-tocop

herol

levelsin

plasma.

Toevaluate

erythrocyte

catalase,SOD

andGPxactiv

ities.

-Erythrocyte

SOD

andcatalase

activ

ities

high

erand

GPxactiv

ities

lower

inPKU

patientsvs

controls.Se

levelsmeasuredin

47.5%

ofthePKUpatientsgrou

p,andwereno

rmal.

Molecular

Genetics

andMetabolism

Phe-restrictedd

diet,with

outfurtherinform

ation

Toevaluate

serum

Phe

levels.

-Plasm

aβ-caroteneandCoQ

10levelslower

inPKU

patientsvs

controls;α-tocop

herollevelssimilarin

thetwogrou

ps.β-Carotene(r=−0

.421

),bu

tno

tCoQ

10,correlated

with

serum

Phe

levelsin

PKU

patients.

Noinform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

n-In

PKUpatients,plasmaTBARS(r=0.70

9)andTA

R(r=−0

.871

),andserum

MDA-LDL(r=0.66

3)levels

correlated

with

serum

Phe

levels.

30age-matched

controls(17–49

y;meanage29

.5±7

.5y)

-In

PKU

patients,erythrocyteGPx(r=−0

.877

),SOD

(r=0.64

7)andcatalase

(r=0.67

2)activ

ities

correlated

with

serum

Phe

levels.

-Serum

NOxlevelshigh

erandADMA

levelsand

ADMA:NOxratio

s(A

DMA/NOx)

lower

inPKU

patientsvs

controls.

-Plasm

aarginine

levelslower

andcitrullin

elevels

high

erin

PKU

grou

pvs

controlgrou

p.

-In

PKU

patients,serum

NOxandADMAvalues,as

wellas

ADMA/NOxratio

s,with

outcorrelationwith

serum

Phe

levels,althou

ghNOxtend

edto

belower

inpatientswith

high

erPhe

levels.


Tab

le1

(con

tinued)

Firstauthor

anddate

/coun

try/journal


n,ageandnu

mber,diet

Aim

/stud

ydesign

Results

(Sittaet

al.20

11)

18classicalPKU

patients,meanage17

.2±2.6y,

rang

e15–2

2y,

underdietarytreatm

ent

(protein-restricteddiet

supp

lementedwith

aspecialform

ulano

tcontaining

LCandSe)

Oxidativ

estress

parameters(plasm

a:TBARS,

proteincarbon

ylandsulfhy

dryl

contents;

erythrocyte:

GSH-Px,

CATandSOD)were

analyzed

inPKU

patientsbefore

andafterat

least6mon

thsof

supp

lementatio

nwith

Se

andLC.To

evaluate

LC

inbloo

dspotsandSein

plasma.

Bothbloo

dLCandplasmaSelevelslower

(p<0.05

)in

PKU

patientsbefore

antio

xidant

supp

lementatio

nvs

controls.Sup

plem

entatio

nreverted

thisdeficiency.

Brazil

Average

bloo

dPhe

levelscalculated

from

the

variou

smeasurementsob

tained

atevery2months¼

686�

315m

mol=L.

Plasm

aTBARShigh

er(p<0.01

)in

PKU

patientsvs

controlsbefore

supp

lementatio

n;supp

lementatio

nreverted

thisprocess.

CellularandMolecular

Neurobiolog

yNoinform

ationprov

ided

regardingdu

ratio

nof

patientsmon

itorizatio

nPlasm

aproteinsulfhy

dryl

grou

pslevelslower

(p<0.01

),bu

tplasmacarbon

ylform

ationhigh

er(p<0.05

),in

PKU

patientswith

outsupp

lementatio

nvs

controls.Sup

plem

entatio

ncorrectedtheox

idation

ofsulfhy

dryl

grou

ps.

18healthycontrolchild

ren,

meanage

19.4±3.7y;

rang

e18–2

3y

Erythrocyte

GSH-Px(p<0.05

)andSOD

(p<0.01

)activ

ities

lower

inPKU

patientsbefore

supp

lemen-

tatio

nvs

controls.Sup

plem

entatio

nincreasedGSH-

Pxactiv

ityto

controlslevel.CATactiv

ityin

PKU

patientssimilarto

controls(beforeandafter

supp

lementatio

n).

Negativecorrelationbetweenbloo

dLClevelsand

plasmaTBARS(r=−0

.560

;p<0.01

)andpo

sitiv

ecorrelationbetweenplasmaSelevelsanderythrocyte

GSH-Pxactiv

ity,in

PKU

patients(r=0.94

5;p<0.01

).

Abb

reviations:8-OHdG

:8-hy

drox

y-2-deox

yguano

sine;ADMA:asym

metricdimethy

larginine;

Apo

AI:apolipop

rotein

AI;CAT:catalase;CoQ

10:coenzymeQ10

;Cu/ZnSOD:copp

erandzinc

superoxide

dism

utase;

Cu:

copp

er;GPX

(GPxandGSH-Px):glutathion

eperoxidase;GR:glutathion

eredu

ctase;

GSH:redu

cedglutathion

e;GST:glutathion

etransferase;

h:ho

ur;HPA

:hy

perpheny

lalaninemia;ID

C:indexof

dietarycontrol;m:mon

th;LC:L-carnitin

e;m-H

PA:mild

-hyp

erph

enylalaninem

ia;MDA:malon

dialdehy

de;MDA-LDL:malon

dialdehy

de-m

odifiedlow-

density

lipop

rotein;NOx:

nitrite/nitrate;

Phe:L-pheny

lalanine;PKU:ph

enylketonu

ria;

PON1:

paraox

onase1;

PON-A

ryl:paraox

onase-arylesterase;Q10

:ub

iquino

ne-10;

Se:

selenium

;SOD:

superoxide

dism

utase;

TAC:totalantio

xidant

capacity;TA

R:totalantio

xidant

reactiv

ity;TA

S:totalantio

xidant

status;TBARS:thiobarbitu

ricacid-reactivespecies;Tyr:tyrosine;vs:versus;y:

year;Zn:

zinc.The

abbreviatio

nsused

inthis

tablematch

thosefrom

therespectiv

earticles.

aIm

possible

todiscriminatebetweenplasmaor

serum,at

somepo

int,in

thearticle.bIm

possible/

difficultto

discriminatebetweenbloo

dor

plasmain

thearticle.cIm

possible

todiscriminatebetweenplasmaor

erythrocyte,

atsomepo

int,in

thearticle


enzyme (PON1). PON1 protects lipids in HDL and LDLagainst oxidation. LDL oxidation favours atherosclerosis andhas been associated with abdominal obesity (Aviram et al.2005; Efrat and Aviram 2010; Florentin et al. 2008; Garin etal. 2005; Holvoet et al. 2008a, b; Précourt et al. 2010; Rizzoet al. 2009; Witztum and Steinberg 2001). Consequently, theclose link between cardiovascular diseases, obesity andoxidative stress (Ando and Fujita 2009; Farbstein et al.2010; Furukawa et al. 2004; Hansel et al. 2006; Holvoet etal. 2008a, b; Hopps et al. 2010) increases the interest in thethree known PONs, considering their importance on tissueand blood oxidative stress control (Aviram et al. 2005;Camps et al. 2009; Getz and Reardon 2004; Ng et al. 2001;Précourt et al. 2010). Just as for LDL oxidation (Sanayamaet al. 2011), there is just one study evaluating PON1 activityin PKU: in the group of patients with higher Phe levels thetotal antioxidant capacity is lower combined with adecreased PON1 activity (Schulpis et al. 2007). We suggestthat future PKU oxidative stress research should includeLDL oxidation and PONs activity evaluation. It is opportuneto emphasize here that the LDL oxidation and PON1 studiesin PKU describe the effects of increased Phe levels on theworsening of oxidative stress parameters and reinforce theneed to maintain a good metabolic control during life span,and not only during the first years of life, in order to preventmental retardation and to protect patients from LDL oxidation(Sanayama et al. 2011; Schulpis et al. 2007). It has beensuggested that increased oxidative stress in PKU patientsolder than 15 years of age can be avoided by keeping theirserum Phe levels below 700–800 μmol/L (Sanayama et al.2011). So, it seems prudent that the evaluation of long termpatient’s risk for oxidative stress should take into consider-

ation their adherence to the diet, keeping in mind thatcompliance usually declines with ageing (Walter and White2004; Walter et al. 2002). Thus, non-adherent patients willprobably have a more severe risk than adherent patients,perhaps similar to that found in the general population wherethe relation between cardiovascular diseases and oxidativestress is well described (Holvoet et al. 2008a, b; Rizzo et al.2009). On the other hand, possibly diet adherent patients willbenefit from the characteristic PKU nutritional ingestion,which may protect them from atherosclerosis (due to theobservation of a less severe serum lipid profile) (Schulpis etal. 2004). Therefore, we would like to underline the need tomaintain PKU patients under treatment and follow-up inorder to allow a detailed description of their health status interms of cardiovascular diseases (Table 2).

Conclusion

Beyond the vegetarian-like diet, potentially rich in anti-oxidants, other aspects, like bad metabolic control andnutritional deficiencies, may modulate the cardiovascularrisk of PKU patients, by interfering with their global redoxstatus. So far, early diagnosed and diet compliant PKUpatients seem to be more protected than late diagnosed andnon-compliant PKU patients, as they present fewer alter-ations of antioxidant defences and less oxidative damage.Future studies, in which more patients at adult ages areincluded and more specific parameters are evaluated, areneeded to help clarifying the influence of the disease and itsdietary treatment on cardiovascular risk. At present, itseems advisable to maintain patients in follow-up, even at

Table 2 Suggestions for clinical monitorization and educational objectives for PKU patients

Clinical follow-up parameters Educational objectives

In blood, annual evaluation of: - To explain that blood lipids (its redox status and antioxidant capacity) areimportant determinants of health, besides Phe.- Lipid profile (total cholesterol, LDL, HDL, TG);

- PON1 activity;

- oxLDL levels;

- TAS.

In diet, annual evaluation of: - To explain the importance of achieving a balanced diet with adequateamounts of all nutrients;- Macro and micronutrient intake from the Phe free protein

substitute and, if possible, from diet natural permitted foods; - To underline that, besides its relevance in PKU metabolic control, Phe-freeprotein substitutes are important sources of crucial macro andmicronutrients;- Vitamin and mineral ingestion from extra supplements;

- To reinforce the role of oxidative stress in determining a good healthstatus, besides Phe.

- Total intake of antioxidant substances, like phytochemicals,Zn, Se, Q10, vitamins C and E.

Annual evaluation of: - To raise awareness that obesity and its consequences may also arise inPKU patients (and can also be related to oxidative stress).- Body mass index and waist circumference;

- Blood pressure.

Abbreviations: HDL: high density lipoprotein; LDL: low density lipoprotein; oxLDL: oxidized low density lipoprotein; Phe: phenylalanine; PKU:phenylketonuria; PON1: paraoxonase 1; Q10: ubiquinone-10; Se: selenium; TAS: total antioxidant status; TG: triglycerides; Zn: zinc


adult ages, with regular monitorization and educationalactivities in order to help them achieve better health.

Acknowledgements The authors gratefully acknowledge FátimaSantos, Isabel Azevedo and Tiago Martins for carefully reading themanuscript.

Details of funding There was no financial support for this article.

Competing interest statement Júlio César Rocha is a member of theMerck Serono European Nutritionist Expert Panel in Phenylketonuria.

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oxidative stress in phenylketonuria: future directions

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